Plasmin variants and uses thereof

ABSTRACT

The invention concerns plasmin variants. Polynucleotides the disclosed plasmin variants are provided. Additionally, methods of using the plasmin polynucleotides and polynucleotides are provided herein.

RELATED APPLICATIONS

The present application claims the benefit of International PatentApplication No. PCT/IB01/01715, filed Aug. 6, 2001 under 35 U.S.C. §119. The present application claims priority, as a divisional a fromU.S. patent application Ser. No. 09/924,340, filed Aug. 6, 2001, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/305,456,filed Jul. 13, 2001; U.S. Provisional Application Ser. No. 60/302,277,filed Jun. 29, 2001; U.S. Provisional Application Ser. No. 60/298,698,filed Jun. 15, 2001; and U.S. Provisional Application Ser. No.60/293,574, filed May 25, 2001, the disclosures of which areincorporated herein by reference in their entiretics.

FIELD OF THE INVENTION

The present invention is directed to GENSET polypeptides, fragmentsthereof, and the regulatory regions located in the 5′- and 3′-ends ofthe genes encoding the polypeptides. The invention also concernspolypeptides encoded by GENSET polynucleotides and fragments thereof.The present invention also relates to recombinant vectors including thepolynucleotides of the present invention, particularly recombinantvectors comprising a GENSET gene regulatory region or a sequenceencoding a GENSET polypeptide, and to host cells containing thepolynucleotides of the invention, as well as to methods of making suchvectors and host cells. The present invention further relates to the useof these recombinant vectors and host cells in the production of thepolypeptides of the invention. The invention further relates toantibodies that specifically bind to the polypeptides of the inventionand to methods for producing such antibodies and fragments thereof. Theinvention also provides for methods of detecting the presence of thepolynucleotides and polypeptides of the present invention in a sample,methods of diagnosis and screening of abnormal GENSET polypeptideexpression and/or biological activity, methods of screening compoundsfor their ability to modulate the activity or expression of the GENSETpolypeptides, and uses of such compounds.

BACKGROUND OF THE INVENTION

cDNAs encoding secreted proteins or fragments thereof represent aparticularly valuable source of therapeutic agents. Thus, there is aneed for the identification and characterization of secreted proteinsand the nucleic acids encoding them.

In addition to being therapeutically useful themselves, secretoryproteins include short peptides, called signal peptides, at their aminotermini which direct their secretion. These signal peptides are encodedby the signal sequences located at the 5′ ends of the coding sequencesof genes encoding secreted proteins. Because these signal peptides willdirect the extracellular secretion of any protein to which they areoperably linked, the signal sequences may be exploited to direct theefficient secretion of any protein by operably linking the signalsequences to a gene encoding the protein for which secretion is desired.In addition, fragments of the signal peptides calledmembrane-translocating sequences may also be used to direct theintracellular import of a peptide or protein of interest. This may provebeneficial in gene therapy strategies in which it is desired to delivera particular gene product to cells other than the cells in which it isproduced. Signal sequences encoding signal peptides also findapplication in simplifying protein purification techniques. In suchapplications, the extracellular secretion of the desired protein greatlyfacilitates purification by reducing the number of undesired proteinsfrom which the desired protein must be selected. Thus, there exists aneed to identify and characterize the 5′ fragments of the genes forsecretory proteins which encode signal peptides.

Sequences coding for secreted proteins may also find application astherapeutics or diagnostics. In particular, such sequences may be usedto determine whether an individual is likely to express a detectablephenotype, such as a disease, as a consequence of a mutation in thecoding sequence for a secreted protein. In instances where theindividual is at risk of suffering from a disease or other undesirablephenotype as a result of a mutation in such a coding sequence, theundesirable phenotype may be corrected by introducing a normal codingsequence using gene therapy. Alternatively, if the undesirable phenotyperesults from overexpression of the protein encoded by the codingsequence, expression of the protein may be reduced using antisense ortriple helix based strategies.

The secreted human polypeptides encoded by the coding sequences may alsobe used as therapeutics by administering them directly to an individualhaving a condition, such as a disease, resulting from a mutation in thesequence encoding the polypeptide. In such an instance, the conditioncan be cured or ameliorated by administering the polypeptide to theindividual.

In addition, the secreted human polypeptides or fragments thereof may beused to generate antibodies useful in determining the tissue type orspecies of origin of a biological sample. The antibodies may also beused to determine the cellular localization of the secreted humanpolypeptides or the cellular localization of polypeptides which havebeen fused to the human polypeptides. In addition, the antibodies mayalso be used in immunoaffinity chromatography techniques to isolate,purify, or enrich the human polypeptide or a target polypeptide whichhas been fused to the human polypeptide.

SUMMARY OF THE INVENTION

The present invention provides a purified or isolated polynucleotidecomprising, consisting of, or consisting essentially of a nucleotidesequence selected from the group consisting of: (a) the sequences of theodd SEQ ID NOs:1-111; (b) the sequences of clone inserts of thedeposited clone pool; (c) the coding sequences of the odd SEQ IDNOs:1-111; (d) the coding sequences of the clone inserts of thedeposited clone pool; (e) the sequences encoding one of the polypeptidesof the even SEQ ID NOs:2-112; (f) the sequences encoding one of thepolypeptides encoded by the clone inserts of the deposited clone pool;(g) the genomic sequences coding for the GENSET polypeptides; (h) the 5′transcriptional regulatory regions of GENSET genes; (i) the 3′transcriptional regulatory regions of GENSET genes; (j) thepolynucleotides comprising the nucleotide sequence of any combination of(g)-(i); (k) the variant polynucleotides of any of the polynucleotidesof (a)-(j); (1) the polynucleotides comprising a nucleotide sequence of(a)-(k), wherein the polynucleotide is single stranded, double stranded,or a portion is single stranded and a portion is double stranded; (m)the polynucleotides comprising a nucleotide sequence complementary toany of the single stranded polynucleotides of (1). The invention furtherprovides for fragments of the nucleic acids and polypeptides of (a)-(m)described above.

Further embodiments of the invention include purified or isolatedpolynucleotides that comprise, consist of, or consist essentially of anucleotide sequence at least 70% identical, more preferably at least75%, and even more preferably at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical, to any of the nucleotide sequences in (a)-(m)above, e.g. over a region of contiguous nucleotides at least about anyone integer between 10 and the last integer representing the lastinteger representing the last nucleotide of a specified sequence of thesequence listing, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide of the present inventionincluding (a) through (m) above.

The present invention also relates to recombinant vectors, which includethe purified or isolated polynucleotides of the present invention, andto host cells recombinant for the polynucleotides of the presentinvention, as well as to methods of making such vectors and host cells.The present invention further relates to the use of these recombinantvectors and recombinant host cells in the production of GENSETpolypeptides. The present invention further relates to a polynucleotideof the present invention operably linked to a regulatory sequenceincluding promoters, enhancers, etc.

The invention further provides a purified or isolated polypeptidecomprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of: (a) the full lengthpolypeptides of even SEQ ID NOs:2-112; (b) the full length polypeptidesencoded by the clone inserts of the deposited clone pool; (c) theepitope-bearing fragments of the polypeptides of even SEQ ID NOs:2-112;(d) the epitope-bearing fragments of the polypeptides encoded by theclone inserts contained in the deposited clone pool; (e) the domains ofthe polypeptides of even SEQ ID NOs:2-112; (f) the domains of thepolypeptides encoded by the clone inserts contained in the depositedclone pool; (g) the signal peptides of the polypeptides of even SEQ IDNOs:2-112 or encoded by the human cDNAs of the deposited clone pool; (h)the mature polypeptides of even SEQ ID Nos:2-112 or encoded by the humancDNAs of the deposited clone pool; and (i) the allelic variantpolypeptides of any of the polypeptides of (a)-(h). The inventionfurther provides for fragments of the polypeptides of (a)-(i) above,such as those having biological activity or comprising biologicallyfunctional domain(s).

The present invention further includes polypeptides with an amino acidsequence with at least 70% similarity, and more preferably at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% 99% similarity to those polypeptidesdescribed in (a)-(i), or fragments thereof, as well as polypeptideshaving an amino acid sequence at least 70% identical, more preferably atleast 75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to those polypeptides described in (a)-(i),or fragments thereof, e.g. over a region of amino acids at least any oneinteger between 6 and the last integer representing the last amino acidof a specified polypeptide sequence of the sequence listing. Theinvention further relates to methods of making the polypeptides of thepresent invention.

The present invention further relates to transgenic plants or animals,wherein said transgenic plant or animal is transgenic for apolynucleotide of the present invention and expresses a polypeptide ofthe present invention.

The invention further relates to antibodies that specifically bind toGENSET polypeptides of the present invention and fragments thereof aswell as to methods for producing such antibodies and fragments thereof.

The invention also provides kits, uses and methods for detecting GENSETgene expression and/or biological activity in a biological sample. Onesuch method involves assaying for the expression of a GENSETpolynucleotide in a biological sample using the polymerase chainreaction (PCR) to amplify and detect GENSET polynucleotides or Southernand Northern blot hybridization to detect GENSET genomic DNA, cDNA ormRNA. Alternatively, a method of detecting GENSET gene expression in atest sample can be accomplished using a compound which binds to a GENSETpolypeptide of the present invention or a portion of a GENSETpolypeptide.

The present invention also relates to diagnostic methods and uses ofGENSET polynucleotides and polypeptides for identifying individuals ornon-human animals having elevated or reduced levels of GENSET geneproducts, which individuals are likely to benefit from therapies tosuppress or enhance GENSET gene expression, respectively, and to methodsof identifying individuals or non-human animals at increased risk fordeveloping, or at present having, certain diseases/disorders associatedwith GENSET polypeptide expression or biological activity.

The present invention also relates to kits, uses and methods ofscreening compounds for their ability to modulate (e.g. increase orinhibit) the activity or expression of GENSET polypeptides includingcompounds that interact with GENSET gene regulatory sequences andcompounds that interact directly or indirectly with a GENSETpolypeptide. Uses of such compounds are also within the scope of thepresent invention.

The present invention also relates to pharmaceutical or physiologicallyacceptable compositions comprising, an active agent, the polypeptides,polynucleotides or antibodies of the present invention, as well as,typically, a physiologically acceptable carrier.

The present invention also relates to computer systems containing cDNAcodes and polypeptide codes of sequences of the invention and tocomputer-related methods of comparing sequences, identifying homology orfeatures using GENSET polypeptides or GENSET polynucleotide sequences ofthe invention.

In another aspect, the present invention provides an isolatedpolynucleotide, the polynucleotide comprising a nucleic acid sequenceencoding a polypeptide of the present invention including thepolypeptide of (a) through (i) above.

In another aspect, the present invention provides a non-human transgenicanimal comprising the host cell.

In another aspect, the present invention provides a method of making aGENSET polypeptide, the method comprising a) providing a population ofhost cells comprising a herein-described polynucleotide and b) culturingthe population of host cells under conditions conducive to theproduction of the polypeptide within said host cells.

In one embodiment, the method further comprises purifying thepolypeptide from the population of host cells.

In another aspect, the present invention provides a method of making aGENSET polypeptide, the method comprising a) providing a population ofcells comprising a polynucleotide encoding a herein-describedpolypeptide; b) culturing the population of cells under conditionsconducive to the production of the polypeptide within the cells; and c)purifying the polypeptide from the population of cells.

In another aspect, the present invention provides a biologically activepolypeptide encoded by any of the herein-described polynucleotides.

In one embodiment, the polypeptide is selectively recognized by anantibody raised against an antigenic polypeptide, or an antigenicfragment thereof, the antigenic polypeptide comprising any one of thesequences shown as even SEQ ID NOs:2-112 or any one of the sequences ofpolypeptides encoded by the human cDNAs of the deposited clone pool.

In another aspect, the present invention provides an antibody thatspecifically binds to any of the herein-described polypeptides andmethods of binding antibody to said polypeptide.

In another aspect, the present invention provides a method ofdetermining whether a GENSET gene is expressed within a mammal, themethod comprising the steps of: a) providing a biological sample fromsaid mammal; b) contacting said biological sample with either of: (i) apolynucleotide that hybridizes under stringent conditions to any of theherein-described polynucleotides; or (ii) a polypeptide thatspecifically binds to any of the herein-described polypeptides; and c)detecting the presence or absence of hybridization between thepolynucleotide and an RNA species within the sample, or the presence orabsence of binding of the polypeptide to a protein within the sample;wherein a detection of the hybridization or of the binding indicatesthat the GENSET gene is expressed within the mammal.

In one embodiment, the polynucleotide is a primer, and the hybridizationis detected by detecting the presence of an amplification productcomprising the sequence of the primer. In another embodiment, thepolypeptide is an antibody.

In another aspect, the present invention provides a method ofdetermining whether a mammal has an elevated or reduced level of GENSETgene expression, the method comprising the steps of: a) providing abiological sample from the mammal; and b) comparing the amount of any ofthe herein-described polypeptides, or of an RNA species encoding thepolypeptide, within the biological sample with a level detected in orexpected from a control sample; wherein an increased amount of thepolypeptide or the RNA species within the biological sample compared tothe level detected in or expected from the control sample indicates thatthe mammal has an elevated level of the GENSET gene expression, andwherein a decreased amount of the polypeptide or the RNA species withinthe biological sample compared to the level detected in or expected fromthe control sample indicates that the mammal has a reduced level of theGENSET gene expression.

In another aspect, the present invention provides a method ofidentifying a candidate modulator of a GENSET polypeptide, the methodcomprising: a) contacting any of the herein-described polypeptides witha test compound; and b) determining whether the compound specificallybinds to the polypeptide; wherein a detection that the compoundspecifically binds to the polypeptide indicates or inhibits or activatesof a specified biological activity that the compound is a candidatemodulator of the GENSET polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram of an exemplary computer system.

FIG. 2 is a flow diagram illustrating one embodiment of a process 200for comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the identity levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 ina computer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence.

BRIEF DESCRIPTION OF TABLES

Table I provides the Applicants' internal designation number (CloneID_Clone Name) which corresponds to each sequence identification number(SEQ ID NO.) of the Sequence Listing, and indicates whether the sequenceis a nucleic acid sequence (DNA) or a polypeptide sequence (PRT).Further provided is information regarding the name of the correspondingnucleic acid or polypeptide sequence, and information regarding thedeposit of biological material. It should he appreciated that biologicalmaterials have been deposited with reference to their correspondingClone ID, Clone Name, or both Clone ID_Clone Name.

Table II provides the positions of the nucleotides of the correspondingSEQ ID NOs. of the Sequence Listing which comprise the open readingframe (ORF), signal peptide, mature peptide, polyadenylation signal, andthe polyA tail of the polynucleotides of the invention.

Table III provides the positions of the amino acid of the correspondingSEQ ID NOs. of the Sequence Listing which comprise the positions ofimmunogenic epitopes of the polypeptides of the invention, which areuseful in antibody generation as described in Example 1.

Table IV provides the positions of the nucleotides comprisingpreferentially included or excluded fragments of the corresponding SEQID NOs. of the Sequence Listing.

BRIEF DESCRIPTION OF SEQUENCES

GENSET polypeptide and polynucleotide sequences are presented in theaccompanying Sequence Listing as SEQ ID NO: 1-112.

Odd SEQ ID NOs:1-111 are the nucleotide sequences of cDNAs, with openreading frames as indicated. When appropriate, the potentialpolyadenylation site and polyadenylation signal are also indicated.

Even SEQ ID NOs:2-112 are the amino acid sequences of proteins encodedby the cDNAs of odd SEQ ID NOs:1-111.

In accordance with the regulations relating to Sequence Listings, thefollowing codes have been used in the Sequence Listing to describesnucleotide sequences. The code “r” in the sequences indicates that thenucleotide may be a guanine or an adenine. The code “y” in the sequencesindicates that the nucleotide may be a thymine or a cytosine. The code“m” in the sequences indicates that the nucleotide may be an adenine ora cytosine. The code “k” in the sequences indicates that the nucleotidemay be a guanine or a thymine. The code “s” in the sequences indicatesthat the nucleotide may be a guanine or a cytosine. The code “w” in thesequences indicates that the nucleotide may be an adenine or an thymine.In addition, all instances of the symbol “n” in the nucleic acidsequences mean that the nucleotide can be adenine, guanine, cytosine orthymine.

In some instances, the polypeptide sequences in the Sequence Listingcontain the symbol “Xaa.” These “Xaa” symbols indicate either (1) aresidue which cannot be identified because of nucleotide sequenceambiguity or (2) a stop codon in the determined sequence whereapplicants believe one should not exist (if the sequence were determinedmore accurately). In some instances, several possible identities of theunknown amino acids may be suggested by the genetic code.

In the case of secreted proteins, it should be noted that, in accordancewith the regulations governing Sequence Listings, in the appendedSequence Listing the encoded protein (i.e. the protein containing thesignal peptide and the mature protein or fragment thereof) extends froman amino acid residue having a negative number through a positivelynumbered amino acid residue. Thus, the first amino acid of the matureprotein resulting from cleavage of the signal peptide is designated asamino acid number 1, and the first amino acid of the signal peptide isdesignated with the appropriate negative number.

In the case that a polynucleotide or polypeptide sequence described inthe specification for SEQ ID NOs:1-112 is in conflict with thecorresponding sequence provided in the Sequence listing, the sequencesprovided in the Sequence listing controls.

It should be appreciated the polynucleotide and polypeptide sequences ofSEQ ID NO:1-112 of the Sequence Listing are hereby incorporated byreference in their entireties.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTSDefinitions

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

The term “GENSET gene,” when used herein, encompasses genomic, mRNA andcDNA sequences encoding a GENSET polypeptide, including the 5′ and 3′untranslated regions of said sequences.

The term “GENSET polypeptide biological activity” or “GENSET biologicalactivity” is intended for polypeptides exhibiting any activity similar,but not necessarily identical, to an activity of a GENSET polypeptide ofthe invention. The GENSET polypeptide biological activity of a givenpolypeptide may be assessed using any suitable biological assay, anumber of which are known to those skilled in the art. In contrast, theterm “biological activity” refers to any activity that any polypeptidemay have.

The term “corresponding mRNA” refers to mRNA which was or can be atemplate for cDNA synthesis for producing a cDNA of the presentinvention.

The term “corresponding genomic DNA” refers to genomic DNA which encodesan mRNA of interest, e.g. corresponding to a cDNA of the invention,which genomic DNA includes the sequence of one of the strands of themRNA, in which thymidine residues in the sequence of the genomic DNA (orcDNA) are replaced by uracil residues in the mRNA.

The term “deposited clone pool” is used herein to refer to the pool ofclones entitled cDNA-11-2000 deposited with the ATCC on Nov. 27, 2000,or cDNA-8-2000, deposited with the ATCC on Sep. 15, 2000.

The term “heterologous”, when used herein, is intended to designate anypolynucleotide or polypeptide other than a GENSET polynucleotide orGENSET polypeptide of the invention, respectively.

“Providing” with respect to, e.g. a biological sample, population ofcells, etc. indicates that the sample, population of cells, etc. issomehow used in a method or procedure. Significantly, “providing” abiological sample or population of cells does not require that thesample or cells are specifically isolated or obtained for the purposesof the invention, but can instead refer, for example, to the use of abiological sample obtained by another individual, for another purpose.

An “amplification product” refers to a product of any amplificationreaction, e.g. PCR, RT-PCR, LCR, etc.

A “modulator” of a protein or other compound refers to any agent thathas a functional effect on the protein, including physical binding tothe protein, alterations of the quantity or quality of expression of theprotein, altering any measurable or detectable activity, property, orbehavior of the protein, or in any way interacts with the protein orcompound. “A test compound” can be any molecule that is evaluated forits ability to modulate a protein or other compound.

An antibody or other compound that specifically binds to a polypeptideor polynucleotide of the invention is also said to “selectivelyrecognize” the polypeptide or polynucleotide.

The term “isolated” with respect to a molecule requires that themolecule be removed from its original environment (e. g., the naturalenvironment if it is naturally occurring). For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or DNA orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotide could be part of avector and/or such polynucleotide or polypeptide could be part of acomposition, and still be isolated in that the vector or composition isnot part of its natural environment. For example, a naturally-occurringpolynucleotide present in a living animal is not isolated, but the samepolynucleotide, separated from some or all of the coexisting materialsin the natural system, is isolated. Specifically excluded from thedefinition of “isolated” are: naturally-occurring chromosomes (such aschromosome spreads), artificial chromosome libraries, genomic libraries,and cDNA libraries that exist either as an in vitro nucleic acidpreparation or as a transfected/transformed host cell preparation,wherein the host cells are either an in vitro heterogeneous preparationor plated as a heterogeneous population of single colonies. Alsospecifically excluded are the above libraries wherein a specifiedpolynucleotide makes up less than 5% (may also be specified as 10%, 25%,50%, or 75%) of the number of nucleic acid inserts in the vectormolecules. Further specifically excluded are whole cell genomic DNA orwhole cell RNA preparations (including said whole cell preparationswhich are mechanically sheared or enzymatically digested). Furtherspecifically excluded are the above whole cell preparations as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis (including blot transfers of the same) wherein thepolynucleotide of the invention has not further been separated from theheterologous polynucleotides in the electrophoresis medium (e.g.,further separating by excising a single band from a heterogeneous bandpopulation in an agarose gel or nylon blot).

The term “purified” does not require absolute purity; rather, it isintended as a relative definition. Purification of starting material ornatural material to at least one order of magnitude, preferably two orthree orders, and more preferably four or five orders of magnitude isexpressly contemplated.

The term “purified” is further used herein to describe a polypeptide orpolynucleotide of the invention which has been separated from othercompounds including, but not limited to, polypeptides orpolynucleotides, carbohydrates, lipids, etc. The term “purified” may beused to specify the separation of monomeric polypeptides of theinvention from oligomeric forms such as homo- or hetero-dimers, trimers,etc. The term “purified” may also be used to specify the separation ofcovalently closed (i.e. circular) polynucleotides from linearpolynucleotides. A substantially pure polypeptide or polynucleotidetypically comprises about 50%, preferably 60 to 90% weight/weight of apolypeptide or polynucleotide sample, respectively, more usually about95%, and preferably is over about 99% pure but, may be specificed as anyinteger of percent between 50 and 100. Polypeptide and polynucleotidepurity, or homogeneity, is indicated by a number of means well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single band upon staining the gel. Forcertain purposes higher resolution can be provided by using HPLC orother means well known in the art. As an alternative embodiment,purification of the polypeptides and polynucleotides of the presentinvention may be expressed as “at least” a percent purity relative toheterologous polypeptides and polynucleotides (DNA, RNA or both). As apreferred embodiment, the polypeptides and polynucleotides of thepresent invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologouspolypeptides and polynucleotides, respectively. As a further preferredembodiment the polypeptides and polynucleotides have a purity rangingfrom any number, to the thousandth position, between 90% and 100% (e.g.,a polypeptide or polynucleotide at least 99.995% pure) relative toeither heterologous polypeptides or polynucleotides, respectively, or asa weight/weight ratio relative to all compounds and molecules other thanthose existing in the carrier. Each number representing a percentpurity, to the thousandth position, may be claimed as individual speciesof purity.

As used interchangeably herein, the terms “nucleic acid molecule(s)”,“oligonucleotide(s)”, and “polynucleotide(s)” include RNA or DNA (eithersingle or double stranded, coding, complementary or antisense), orRNA/DNA hybrid sequences of more than one nucleotide in either singlechain or duplex form (although each of the above species may beparticularly specified). The term “nucleotide” is used herein as anadjective to describe molecules comprising RNA, DNA, or RNA/DNA hybridsequences of any length in single-stranded or duplex form. Moreprecisely, the expression “nucleotide sequence” encompasses the nucleicmaterial itself and is thus not restricted to the sequence information(i.e. the succession of letters chosen among the four base letters) thatbiochemically characterizes a specific DNA or RNA molecule. The term“nucleotide” is also used herein as a noun to refer to individualnucleotides or varieties of nucleotides, meaning a molecule, orindividual unit in a larger nucleic acid molecule, comprising a purineor pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphategroup, or phosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. The term “nucleotide” is also usedherein to encompass “modified nucleotides” which comprise at least onemodification such as (a) an alternative linking group, (b) an analogousform of purine, (c) an analogous form of pyrimidine, or (d) an analogoussugar. For examples of analogous linking groups, purine, pyrimidines,and sugars, see, for example, PCT publication No. WO 95/04064, whichdisclosure is hereby incorporated by reference in its entirety.Preferred modifications of the present invention include, but are notlimited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v)ybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and2,6-diaminopurine. The polynucleotide sequences of the invention may beprepared by any known method, including synthetic, recombinant, ex vivogeneration, or a combination thereof, as well as utilizing anypurification methods known in the art. Methylenemethylimino linkedoligonucleosides as well as mixed backbone compounds having, may beprepared as described in U.S. Pat. Nos. 5,378,825; 5,386,023; 5,489,677;5,602,240; and 5,610,289, which disclosures are hereby incorporated byreference in their entireties. Formacetal and thioformacetal linkedoligonucleosides may be prepared as described in U.S. Pat. Nos.5,264,562 and 5,264,564, which disclosures are hereby incorporated byreference in their entireties. Ethylene oxide linked oligonucleosidesmay be prepared as described in U.S. Pat. No. 5,223,618, whichdisclosure is hereby incorporated by reference in its entirety.Phosphinate oligonucleotides may be prepared as described in U.S. Pat.No. 5,508,270, which disclosure is hereby incorporated by reference inits entirety. Alkyl phosphonate oligonucleotides may be prepared asdescribed in U.S. Pat. No. 4,469,863, which disclosure is herebyincorporated by reference in its entirety. 3′-Deoxy-3′-methylenephosphonate oligonucleotides may be prepared as described in U.S. Pat.Nos. 5,610,289 or 5,625,050 which disclosures are hereby incorporated byreference in their entireties. Phosphoramidite oligonucleotides may beprepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No.5,366,878 which disclosures are hereby incorporated by reference intheir entireties. Alkylphosphonothioate oligonucleotides may be preparedas described in published PCT applications WO 94/17093 and WO 94/02499which disclosures are hereby incorporated by reference in theirentireties. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides may beprepared as described in U.S. Pat. No. 5,476,925, which disclosure ishereby incorporated by reference in its entirety. Phosphotriesteroligonucleotides may be prepared as described in U.S. Pat. No.5,023,243, which disclosure is hereby incorporated by reference in itsentirety. Borano phosphate oligonucleotides may be prepared as describedin U.S. Pat. Nos. 5,130,302 and 5,177,198 which disclosures are herebyincorporated by reference in their entireties.

The term “upstream” is used herein to refer to a location which istoward the 5′ end of the polynucleotide from a specific reference point.

The terms “base paired” and “Watson & Crick base paired” are usedinterchangeably herein to refer to nucleotides which can be hydrogenbonded to one another by virtue of their sequence identities in a mannerlike that found in double-helical DNA with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds (see Stryer, (1995)Biochemistry, 4th edition, which disclosure is hereby incorporated byreference in its entirety).

The terms “complementary” or “complement thereof” are used herein torefer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. For the purpose ofthe present invention, a first polynucleotide is deemed to becomplementary to a second polynucleotide when each base in the firstpolynucleotide is paired with its complementary base. Complementarybases are, generally, A and T (or A and U), or C and G. “Complement” isused herein as a synonym from “complementary polynucleotide”,“complementary nucleic acid” and “complementary nucleotide sequence”.These terms are applied to pairs of polynucleotides based solely upontheir sequences and not any particular set of conditions under which thetwo polynucleotides would actually bind. Unless otherwise stated, allcomplementary polynucleotides are fully complementary on the wholelength of the considered polynucleotide.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymer of amino acids without regard to the length of thepolymer; thus, peptides, oligopeptides, and proteins are included withinthe definition of polypeptide. This term also does not specify orexclude chemical or post-expression modifications of the polypeptides ofthe invention, although chemical or post-expression modifications ofthese polypeptides may be included or excluded as specific embodiments.Therefore, for example, modifications to polypeptides that include thecovalent attachment of glycosyl groups, acetyl groups, phosphate groups,lipid groups and the like are expressly encompassed by the termpolypeptide. Further, polypeptides with these modifications may bespecified as individual species to be included or excluded from thepresent invention. The natural or other chemical modifications, such asthose listed in examples above can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Polypeptides may be branched, for example, as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.[See, for instance Creighton, (1993), Posttranslational CovalentModification of Proteins, W.H. Freeman and Company, New York B. C.Johnson, Ed., Academic Press, New York 1-12; Seifter, et al., (1990)Meth Enzymol 182:626-646; Rattan et al., (1992) Ann NY Acad Sci663:48-62]. Also included within the definition are polypeptides whichcontain one or more analogs of an amino acid (including, for example,non-naturally occurring amino acids, amino acids which only occurnaturally in an unrelated biological system, modified amino acids frommammalian systems, etc.), polypeptides with substituted linkages, aswell as other modifications known in the art, both naturally occurringand non-naturally occurring.

As used herein, the terms “recombinant polynucleotide” and“polynucleotide construct” are used interchangeably to refer to linearor circular, purified or isolated polynucleotides that have beenartificially designed and which comprise at least two nucleotidesequences that are not found as contiguous nucleotide sequences in theirinitial natural environment. In particular, these terms mean that thepolynucleotide or cDNA is adjacent to “backbone” nucleic acid to whichit is not adjacent in its natural environment. Additionally, to be“enriched” the cDNAs will represent 5% or more of the number of nucleicacid inserts in a population of nucleic acid backbone molecules.Backbone molecules according to the present invention include nucleicacids such as expression vectors, self-replicating nucleic acids,viruses, integrating nucleic acids, and other vectors or nucleic acidsused to maintain or manipulate a nucleic acid insert of interest.Preferably, the enriched cDNAs represent 15% or more of the number ofnucleic acid inserts in the population of recombinant backbonemolecules. More preferably, the enriched cDNAs represent 50% or more ofthe number of nucleic acid inserts in the population of recombinantbackbone molecules. In a highly preferred embodiment, the enriched cDNAsrepresent 90% or more (including any number between 90 and 100%, to thethousandth position, e.g., 99.5%) of the number of nucleic acid insertsin the population of recombinant backbone molecules.

The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A sequence whichis “operably linked” to a regulatory sequence such as a promoter meansthat said regulatory element is in the correct location and orientationin relation to the nucleic acid to control RNA polymerase initiation andexpression of the nucleic acid of interest. For instance, a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the coding sequence.

The term “domain” refers to an amino acid fragment with specificbiological properties. This term encompasses all known structural andlinear biological motifs. Examples of such motifs include but are notlimited to leucine zippers, helix-turn-helix motifs, glycosylationsites, ubiquitination sites, alpha helices, and beta sheets, signalpeptides which direct the secretion of proteins, sites forpost-translational modification, enzymatic active sites, substratebinding sites, and enzymatic cleavage sites.

Although each of these terms has a distinct meaning, the terms“comprising”, “consisting of” and “consisting essentially of” may beinterchanged for one another throughout the instant application. Theterm “having” has the same meaning as “comprising” and may be replacedwith either the term “consisting of” or “consisting essentially of”.

Unless otherwise specified in the application, nucleotides and aminoacids of polynucleotides and polypeptides, respectively, of the presentinvention are contiguous and not interrupted by heterologous sequences.

The term “neoplastic cells” as used herein refers to cells that resultfrom abnormal new growth. A neoplastic cell further includes transformedcells, cancer cells including blood cancers and solid tumors (benign andmalignant).

As used herein, the term “tumor” refers to an abnormal mass orpopulation of cells that result from excessive cell division, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “tumor” is further defined as two or more neoplastic cells.

“Malignant tumors” are distinguished from benign growths or tumors inthat, in addition to uncontrolled cellular proliferation, they willinvade surrounding tissues and may additionally metastasize.

The term “transformed cells,” “malignant cells” or “cancer” areinterchangeable and refer to cells that have undergone malignanttransformation, but may also include lymphocyte cells that haveundergone blast transformation. Malignant transformation is a conversionof normal cells to malignant cells. Transformed cells have a greaterability to cause tumors when injected into animals. Transformation canbe recognized by changes in growth characteristics, particularly inrequirements for macromolecular growth factors, and often also bychanges in morphology. Transformed cells usually proliferate withoutrequiring adhesion to a substratum and usually lack cell to cellinhibition and pile up after forming a monolayer in cell culture.

The term “neoplastic disease” as used herein refers to a conditioncharacterized by uncontrolled, abnormal growth of cells. Neoplasticdiseases include cancer. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, ovarian cancer, cervical cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer,bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer,endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, braincancer, ovarian cancer, neuroblastoma, myeloma, various types of headand neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia,Ewing sarcoma and peripheral neuroepithelioma. All of the possiblecancers listed herein are included in, or may be excluded from, thepresent invention as individual species.

As used herein, the term “carcinoma” refers to a new growth that arisesfrom epithelium, found in skin or, more commonly, the lining of bodyorgans (adenocarcinoma), for example: breast, prostate, lung, stomach orbowel. Carcinomas include bladder carcinoma, hepatocarcinoma,hepatoblastoma, rhabdomyosarcoma, ovarian carcinoma, cervical carcinoma,lung carcinoma, breast carcinoma, colorectal carcinoma, uterine cervicalcancer carcinoma, endometrioid carcinoma, paraganglioma, squamous cellcarcinoma in head and neck, esophageal carcinoma, thyroid carcinoma,astrocytoma, neuroblastoma and neuroepithelioma. All of the possiblecarcinomas listed herein are included in, or may be excluded from, thepresent invention as individual species.

The term “immortalized cells” as used herein refers to cells reproduceindefinitely. The cells escape from the normal limitation on growth of afinite number of division cycles. The term does not include malignantcells.

The term “normal cells” as used herein refers to cells that have alimitation on growth, i.e. a finite number of division cycles (theHayflick limit); therefore, is a nontumorigenic cell. Normal cellinclude primary cells, which is a cell or cell line taken directly froma living organism which is not immortalized.

The term “cell cycle” as used herein refers to the cyclic biochemicaland structural events occurring during growth and division of cells. Thestages of the cell cycle include G₀ (Gap 0; rest phase), G1 (Gap 1), Sphase (DNA synthesis), G2 (Gap 2) and M phase (mitosis).

The term “cell growth” as used herein refers to an increase in the sizeof a population of cells.

The term “cell division” as used herein refers to mitosis, i.e., theprocess of cell reproduction.

The term “proliferation” as used herein means growth and division ofcells. “Actively proliferating” means cells that are actively growingand dividing.

The term “inhibiting cellular proliferation” as used herein refers toslowing and/or preventing the growth and division of cells. Cells mayfurther be specified as being arrested in a particular cell cycle stage:G1 (Gap 1), S phase (DNA synthesis), G2 (Gap 2) or M phase (mitosis).

The term “preferentially inhibiting cellular proliferation” as usedherein refers to slowing and/or preventing the growth and division ofcells as compared to normal cells.

The term “metastasis” refers to the transfer of disease (e.g., cancer)from one organ and/or tissue to another not directly connected with it.As used herein, metastasis refers to neoplastic cell growth in anunregulated fashion and spread to distal tissues and organs of the body.

The term “inhibiting metastasis” refers to slowing and/or preventingmetastasis or the spread of neoplastic cells to a site remote from theprimary growth area.

The term “invasion” as used herein refers to the spread of cancerouscells to surrounding tissues.

The term “inhibiting invasion” refers to slowing and/or preventing thespread of cancerous cells to surrounding tissues.

The term “apoptosis” as used herein refers to programmed cell death assignaled by the nuclei in normally functioning human and animal cellswhen age or state of cell health and condition dictates. “Apoptosis” isan active process requiring metabolic activity by the dying cell, oftencharacterized by cleavage of the DNA into fragments that give a socalled laddering pattern on gels. Cells that die by apoptosis do notusually elicit the inflammatory responses that are associated withnecrosis, though the reasons are not clear. Cancerous cells, however,are unable to experience, or have a reduction in, the normal celltransduction or apoptosis-driven natural cell death process.Morphologically, apoptosis is characterized by loss of contact withneighboring cells, concentration of cytoplasm, endonucleaseactivity-associated chromatin condensation and pyknosis, andsegmentation of the nucleus, among others.

The term “necrosis” as used herein refers to the sum of themorphological changes indicative of cell death and caused by theprogressive degradative action of enzymes, it may affect groups of cellsor part of a structure or an organ. Morphologically, necrosis ischaracterized by marked swelling of mitochondria, swelling of cytoplasmand nuclear alteration, followed by cell destruction and autolysis. Itoccurs passively or incidentally.

The term “inducing apoptosis” refers to increasing the number of cellsthat undergo apoptosis, or the rate by which cells undergo apoptosis, ina given cell population. Preferably the increase is at least 1.25, 1.5,2, 5, 10, 50, 100, 500 or 1000 fold increase as compared to normal,untreated or negative control cells.

The term “inhibiting apoptosis” refers to any decrease in the number ofcells which undergo apoptosis relative to an untreated control.Preferably, the decrease is at least 1.25, 1.5, 2, 5, 10, 50, 100, 500or 1000 fold decrease as compared to normal, untreated or negativecontrol cells.

An “effective amount” of a composition disclosed herein or an agonistthereof, in reference to “inhibiting the cellular proliferation” of aneoplastic cell, is an amount capable of inhibiting, to some extent, thegrowth of target cells. The term further includes an amount capable ofinvoking a growth inhibitory, cytostatic and/or cytotoxic effect and/orapoptosis and/or necrosis of the target cells. An “effective amount” ofa polypeptide of the present invention or an agonist thereof forpurposes of inhibiting neoplastic cell growth may be determinedempirically and in a routine manner using methods well known in the art.

A “therapeutically effective amount”, in reference to the treatment ofneoplastic disease or neoplastic cells, refers to an amount capable ofinvoking one or more of the following effects: (1) inhibition, to someextent, of tumor growth, including, (i) slowing down and (ii) completegrowth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion or (v) reducing, slowing or preventing metastasis; and/or (8)relief, to some extent, of one or more symptoms associated with thedisorder. A “therapeutically effective amount” of a polypeptide of thepresent invention or an agonist thereof for purposes of treatment oftumor may be determined empirically and in a routine manner.

A “growth inhibitory amount” of a Polypeptide of the present inventionor an agonist thereof is an amount capable of inhibiting the growth of acell, especially a malignant tumor cell, e.g., cancer cell, either invitro or in vivo. A “growth inhibitory amount” of a polypeptide of thepresent invention or an agonist thereof for purposes of inhibitingneoplastic cell growth may be determined empirically and in a routinemanner using methods well known in the art.

A “cytotoxic amount” of a polypeptide of the present invention or anagonist thereof is an amount capable of causing the destruction of acell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A“cytotoxic amount” of a polypeptide of the present invention or anagonist thereof for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner using methods well knownin the art.

The terms “killing” or “inducing cytotoxicity” as used herein refer toinducing cell death by either apoptosis and/or necrosis, wherebyembodiments of the invention include only apoptosis, only necrosis andboth apoptosis and necrosis.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells, for example by inhibitingprogression of the cell cycle, and/or causes cell death. The term isintended to include radioactive isotopes, chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disease or condition so asto prevent a physical manifestation of the disease or condition.Alternatively, the term “preventing” can also be used to signify thereduction, or severity, of clinical symptoms associated with a diseaseor condition.

“Suppression” involves administration of drug prior to the clinicalappearance of disease.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms.

In human and veterinary medicine, we use the term “prophylaxis” asdistinct from “treatment” to encompass “preventing” and “suppressing”.Herein, “protection” includes “prophylaxis”. Protection need not beabsolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, etc in thecase of humans; veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

The term “perceives a need for treatment” refers to a sub-clinicaldetermination that an individual desires treatment. The term “perceivesa need for treatment” in other embodiments can refer to the decisionthat an owner of an animal makes for treatment of the animal.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

As used herein, the term “non-human animal” refers to any non-humananimal, including insects, birds, rodents and more usually mammals.Preferred non-human animals include: primates; farm animals such asswine, goats, sheep, donkeys, cattle, horses, chickens, rabbits; androdents, preferably rats or mice. As used herein, the term “animal” isused to refer to any species in the animal kingdom, preferablyvertebrates, including birds and fish, and more preferable a mammal.Both the terms “animal” and “mammal” expressly embrace human subjectsunless preceded with the term “non-human”. As used herein, the terms“physiologically acceptable,” “pharmaceutically acceptable,” and“pharmaceutical” are interchangeable.

Identity Between Nucleic Acids or Polypeptides

The terms “percentage of sequence identity” and “percentage homology”are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Identity is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, CLUSTALW, FASTDB [Pearson and Lipman, (1988), Proc. Natl.Acad. Sci. USA 85(8):2444-2448; Altschul et al., (1990), J. Mol. Biol.215(3):403-410; Thompson et al. (1994), Nucleic Acids Res.22(2):4673-4680; Higgins et al., (1996), Meth. Enzymol. 266:383-402;Altschul et al., (1993), Nature Genetics 3:266-272; Brutlag et al.(1990) Comp. App. Biosci. 6:237-24], the disclosures of which areincorporated by reference in their entireties.

In a particularly preferred embodiment, protein and nucleic acidsequence identities are evaluated using the Basic Local Alignment SearchTool (“BLAST”) which is well known in the art [e.g., Karlin andAltschul, (1990), Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul etal., (1997), Nuc. Acids Res. 25:3389-3402] the disclosures of which areincorporated by reference in their entireties. In particular, fivespecific BLAST programs are used to perform the following task:

-   -   (1) LASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) LASTX compares the six-frame conceptual translation products        of a query nucleotide sequence (both strands) against a protein        sequence database;    -   (4) BLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) BLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix [Gonnet etal., (1992), Science 256:1443-1445; Henikoff and Henikoff, (1993),Proteins 17:49-61, the disclosures of which are incorporated byreference in their entireties]. Less preferably, the PAM or PAM250matrices may also be used [see, e.g., Schwartz and Dayhoff, (1978),eds., Matrices for Detecting Distance Relationships: Atlas of ProteinSequence and Structure, Washington: National Biomedical ResearchFoundation, the disclosure of which is incorporated by reference in itsentirety]. The BLAST programs evaluate the statistical significance ofall high-scoring segment pairs identified, and preferably selects thosesegments which satisfy a user-specified threshold of significance, suchas a user-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (see, e.g., Karlin andAltschul, 1990), the disclosure of which is incorporated by reference inits entirety. The BLAST programs may be used with the default parametersor with modified parameters provided by the user.

Another preferred method for determining the best overall match betweena query nucleotide sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (1990), the disclosure of which is incorporated byreference in its entirety. In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byfirst converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. If the subject sequence is shorter thanthe query sequence because of 5′ or 3′ deletions, not because ofinternal deletions, a manual correction must be made to the results.This is because the FASTDB program does not account for 5′ and 3′truncations of the subject sequence when calculating percent identity.For subject sequences truncated at the 5′ or 3′ ends, relative to thequery sequence, the percent identity is corrected by calculating thenumber of bases of the query sequence that are 5′ and 3′ of the subjectsequence, which are not matched/aligned, as a percent of the total basesof the query sequence. Whether a nucleotide is matched/aligned isdetermined by results of the FASTDB sequence alignment. This percentageis then subtracted from the percent identity, calculated by the aboveFASTDB program using 10, the specified parameters, to arrive at a finalpercent identity score. This corrected score is what is used for thepurposes of the present invention. Only nucleotides outside the 5′ and3′ nucleotides of the subject sequence, as displayed by the FASTDBalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore. For example, a 90 nucleotide subject sequence is aligned to a 100nucleotide query sequence to determine percent identity. The deletionsoccur at the 5′ end of the subject sequence and therefore, the FASTDBalignment does not show a matched/alignment of the first 10 nucleotidesat 5′ end. The 10 unpaired nucleotides represent 10% of the sequence(number of nucleotides at the 5′ and 3′ ends not matched/total number ofnucleotides in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90nucleotides were perfectly matched the final percent identity would be90%. In another example, a 90 nucleotide subject sequence is comparedwith a 100 nucleotide query sequence. This time the deletions areinternal deletions so that there are no nucleotides on the 5′ or 3′ ofthe subject sequence which are not matched/aligned with the query. Inthis case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected. No other manual corrections are made for thepurposes of the present invention. Another preferred method fordetermining the best overall match between a query amino acid sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (1990).In a sequence alignment the query and subject sequences are both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. If the subject sequence is shorter than the query sequence dueto N-or C-terminal deletions, not because of internal deletions, theresults, in percent identity, must be manually corrected. This isbecause the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. Whether a residue is matched/aligned is determined byresults of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This final percent identity score is what is used forthe purposes of the present invention. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query amino acidresidues outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100-residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not match/align with the firstresidues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90-residue subjectsequence is compared with a 100-residue query sequence. This time thedeletions are internal so there are no residues at the N- or C-terminiof the subject sequence, which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only residue positions outside the N- andC-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected. No other manual corrections are made for thepurposes of the present invention.

The term “percentage of sequence similarity” refers to comparisonsbetween polypeptide sequences and is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which an identical or equivalentamino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison and multiplying theresult by 100 to yield the percentage of sequence similarity. Similarityis evaluated using any of the variety of sequence comparison algorithmsand programs known in the art, including those described above in thissection. Equivalent amino acid residues are defined herein in the“Mutated polypeptides” section.

Polynucleotides of the Invention

The present invention concerns GENSET genomic and cDNA sequences. Thepresent invention encompasses GENSET genes, polynucleotides comprisingGENSET genomic and cDNA sequences, as well as fragments and variantsthereof. These polynucleotides may be purified, isolated, orrecombinant.

Also encompassed by the present invention are allelic variants,orthologs, splice variants, and/or species homologues of the GENSETgenes. Procedures known in the art can be used to obtain full-lengthgenes and cDNAs, allelic variants, splice variants, full-length codingportions, orthologs, and/or species homologues of genes and cDNAscorresponding to a nucleotide sequence selected from the groupconsisting of sequences of odd SEQ ID NOs:1-111 and sequences of cloneinserts of the deposited clone pool, using information from thesequences disclosed herein or the clone pool deposited with the ATCC.For example, allelic variants, orthologs and/or species homologues maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue using any techniqueknown to those skilled in the art including those described into thesection entitled “To find similar sequences”.

In a specific embodiment, the polynucleotides of the invention are atleast 15, 30, 50, 100, 125, 500, or 1000 continuous nucleotides. Inanother embodiment, the polynucleotides are less than or equal to 300kb, 200 kb, 100 kb, 50 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2 kb, 1.5 kb, or1 kb in length. In a further embodiment, polynucleotides of theinvention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1naturally occurring genomic flanking gene(s).

Deposited Clone Pool of the Invention

Expression of GENSET genes has been shown to lead to the production ofat least one mRNA species per GENSET gene, which cDNA sequence is setforth in the appended Sequence Listing as odd SEQ ID NOs:1-111. ThecDNAs corresponding to these GENSET mRNA species were cloned either inthe vector pBluescriptII SK⁻ (Stratagene) or in a vector called pPT.Cells containing the cloned cDNAs of the present invention aremaintained in permanent deposit by the inventors at Genset, S.A., 24 RueRoyale, 75008 Paris, France. Table I provides Genset's internaldesignation number assigned to each SEQ ID NO., and indicates whetherthe sequence is a nucleic acid sequence (DNA) or a protein (PRT)sequence. Each cDNA can be removed from the Bluescript vector in whichit was inserted by performing a NotI PstI double digestion, or from thepPT vector by performing a MunI HindIII double digestion, to produce theappropriate fragment for each clone, provided the cDNA sequence does notcontain any of the corresponding restriction sites within its sequence.Alternatively, other restriction enzymes of the multicloning site of thevector may be used to recover the desired insert as indicated by themanufacturer.

Pools of cells containing GENSET genes as described in the SequenceListing, from which the cells containing a particular polynucleotide isobtainable, were or will be also deposited with the American TissueCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, United States. Each cDNA clone has been transfected intoseparate bacterial cells (E-coli) for these composite deposits.

Bacterial cells containing a particular clone can be obtained from thecomposite deposit as follows:

-   -   An oligonucleotide probe or probes should be designed to the        sequence that is known for that particular clone. This sequence        can be derived from the sequences provided herein, or from a        combination of those sequences. The design of the        oligonucleotide probe should preferably follow these parameters:        -   (a) it should be designed to an area of the sequence which            has the fewest ambiguous bases (“N's”), if any;        -   (b) preferably, the probe is designed to have a Tm of            approximately 80 degrees Celsius (assuming 2 degrees for            each A or T and 4 degrees for each G or C). However, probes            having melting temperatures between 40 degrees Celsius and            80 degrees Celsius may also be used provided that            specificity is not lost.

The oligonucleotide should preferably be labeled with gamma[³²P]ATP(specific activity 6000 Ci/mmole) and T4 polynucleotide kinase usingcommonly employed techniques for labeling oligonucleotides. Otherlabeling techniques can also be used. Unincorporated label shouldpreferably be removed by gel filtration chromatography or otherestablished methods. The amount of radioactivity incorporated into theprobe should be quantified by measurement in a scintillation counter.Preferably, specific activity of the resulting probe should beapproximately 4×10⁶ dpm/pmole.

The bacterial culture containing the pool of full-length clones shouldpreferably be thawed and 100 ul of the stock used to inoculate a sterileculture flask containing 25 ml of sterile L-broth containing ampicillinat 100 ug/ml. The culture should preferably be grown to saturation at 37degrees Celsius, and the saturated culture should preferably be dilutedin fresh L-broth. Aliquots of these dilutions should preferably beplated to determine the dilution and volume which will yieldapproximately 5000 distinct and well-separated colonies on solidbacteriological media containing L-broth containing ampicillin at 100ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37degrees Celsius. Other known methods of obtaining distinct,well-separated colonies can also be employed.

Standard colony hybridization procedures should then be used to transferthe colonies to nitrocellulose filters and lyse, denature and bake them.

The filter is then preferably incubated at 65 degrees Celsius for 1 hourwith gentle agitation in 6×SSC (20× stock is 175.3 g NaCl/liter, 88.2 gNa citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100pg/ml of yeast RNA, and 10 mM EDTA (approximately 10 ml per 150 mmfilter). Preferably, the probe is then added to the hybridization mix ata concentration greater than or equal to 1×10⁶ dpm/ml. The filter isthen preferably incubated at 65 degrees Celsius with gentle agitationovernight. The filter is then preferably washed in 500 ml of 2×SSC/0.1%SDS at room temperature with gentle shaking for 15 minutes. A third washwith 0.1×SSC/0.5% SDS at 65 degrees Celsius for 30 minutes to 1 hour isoptional. The filter is then preferably dried and subjected toautoradiography for sufficient time to visualize the positives on theX-ray film. Other known hybridization methods can also be employed.

The positive colonies are picked, grown in culture, and plasmid DNAisolated using standard procedures. The clones can then be verified byrestriction analysis, hybridization analysis, or DNA sequencing. Theplasmid DNA obtained using these procedures may then be manipulatedusing standard cloning techniques familiar to those skilled in the art.

Alternatively, to recover cDNA inserts from the pool of bacteria, a PCRcan be performed on plasmid DNA isolated using standard procedures andprimers designed at both ends of the cDNA insertion, including primersdesigned in the multicloning site of the vector. If a specific cDNA ofinterest is to be recovered, primers may be designed in order to bespecific for the 5′ end and the 3′ end of this cDNA using sequenceinformation available from the appended sequence listing. The PCRproduct which corresponds to the cDNA of interest can then bemanipulated using standard cloning techniques familiar to those skilledin the art.

Therefore, an object of the invention is an isolated, purified, orrecombinant polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of human cDNA inserts of the deposited clonepool. Moreover, preferred polynucleotides of the invention includepurified, isolated, or recombinant GENSET cDNAs consisting of,consisting essentially of, or comprising a nucleotide sequence selectedfrom the group consisting of human cDNA inserts of the deposited clonepool.

cDNA Sequences of the Invention

Another object of the invention is a purified, isolated, or recombinantpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of the polynucleotide sequences of the appended SequenceListing, the sequences of human cDNA clone inserts of the depositedclone pool, complementary sequences thereto, and fragments thereof.Moreover, preferred polynucleotides of the invention include purified,isolated, or recombinant GENSET cDNAs consisting of, consistingessentially of, or comprising a sequence selected from the groupconsisting of the polynucleotide sequences of the Sequence Listing andthe sequences of clone inserts of the deposited clone pool. Structuralparameters of each of the cDNAs of the present invention are presentedin the appended Sequence Listing. Accordingly, the coding sequence (CDS)or open reading frame (ORF) of each cDNA of the invention refers to thenucleotide sequence beginning with the first nucleotide of the startcodon and ending with the last nucleotide of the stop codon. Similarly,the 5′ untranslated region (or 5′ UTR) of each cDNA of the inventionrefers to the nucleotide sequence starting at nucleotide 1 and ending atthe nucleotide immediately 5′ to the first nucleotide of the startcodon. The 3′ untranslated region (or 3′ UTR) of each cDNA of theinvention refers to the nucleotide sequence starting at the nucleotideimmediately 3′ to the last nucleotide of the stop codon and ending atthe last nucleotide of the cDNA.

Untranslated Regions

In addition, the invention concerns a purified, isolated, andrecombinant nucleic acid comprising a nucleotide sequence selected fromthe group consisting of the 5′ UTRs of the polynucleotide sequences ofthe appended Sequence Listing, those of human cDNA clone inserts of thedeposited clone pool, sequences complementary thereto, and allelicvariants thereof. The invention also concerns a purified, isolated,and/or recombinant nucleic acid comprising a nucleotide sequenceselected from the group consisting of the 3′ UTRs of the polynucleotidesequences of the appended Sequence Listing, those of human cDNA cloneinserts of the deposited clone pool, sequences complementary thereto,and allelic variants thereof.

These polynucleotides may be used to detect the presence of GENSET mRNAspecies in a biological sample using either hybridization or RT-PCRtechniques well known to those skilled in the art.

In addition, these polynucleotides may be used as regulatory moleculesable to affect the processing and maturation of any polynucleotideincluding them (either a GENSET polynucleotide or an heterologouspolynucleotide), preferably the localization, stability and/ortranslation of said polynucleotide including them [for a review on UTRssee Decker and Parker, (1995) Curr. Opin. Cell. Biol. 7(3):368-92,Derrigo et al., (2000) Int. J. Mol. Med. 5(2):111-23]. In particular 3′UTRs may be used in order to control the stability of heterologous mRNAsin recombinant vectors using any methods known to those skilled in theart including Makrides (1999) Protein Expr Purif 1999November;17(2):183-202), U.S. Pat. Nos. 5,925,564; 5,807,707 and5,756,264, which disclosures are hereby incorporated by reference intheir entireties.

Coding Sequences

Another object of the invention is an isolated, purified or recombinantpolynucleotide comprising the coding sequence of a sequence selectedfrom the group consisting of the polynucleotide sequences of theappended Sequence Listing, those of human cDNA clone inserts of thedeposited clone pool and variants thereof.

A further object of the invention is an isolated, purified, orrecombinant polynucleotide encoding a polypeptide of the presentinvention.

It will be appreciated that should the extent of the coding sequencediffer from that indicated in the appended sequence listing as a resultof a sequencing error, reverse transcription or amplification error,mRNA splicing, post-translational modification of the encoded protein,enzymatic cleavage of the encoded protein, or other biological factors,one skilled in the art would be readily able to identify the extent ofthe coding sequences in the polynucleotide sequences of the SequenceListing, those of the human cDNA inserts of the deposited clone pool,and allelic variants thereof. Accordingly, the scope of any claimsherein relating to nucleic acids containing the coding sequence of oneof the polynucleotide sequences of the Sequence Listing and those of thecDNA inserts of the deposited clone pool is not to be construed asexcluding any readily identifiable variations from or equivalents to thecoding sequences described in the appended sequence listing. Equivalentsinclude any alterations in a nucleotide coding sequence that does notresult in an amino acid change, or that results in a conservative aminoacid substitution, as defined below, in the polypeptide encoded by thenucleotide sequence. Similarly, should the extent of the polypeptidesdiffer from those indicated in the appended Sequence Listing as a resultof any of the preceding factors, the scope of claims relating topolypeptides comprising the amino acid sequence of the polypeptidesequences of the appended Sequence Listing is not to be construed asexcluding any readily identifiable variations from or equivalents to thesequences described in the appended sequence listing.

The above disclosed polynucleotides that contain the coding sequence ofthe GENSET genes may be expressed in a desired host cell or a desiredhost organism, when this polynucleotide is placed under the control ofsuitable expression signals. The expression signals may be either theexpression signals contained in the regulatory regions in the GENSETgenes of the invention or, in contrast, the signals may be exogenousregulatory nucleic sequences. Such a polynucleotide, when placed underthe suitable expression signals, may also be inserted in a vector forits expression and/or amplification. Further included in the presentinvention are polynucleotides encoding the polypeptides of the presentinvention that are fused in frame to the coding sequences for additionalheterologous amino acid sequences. Also included in the presentinvention are nucleic acids encoding polypeptides of the presentinvention together with additional, non-coding sequences, including, butnot limited to, non-coding 5′ and 3′ sequences, vector sequence,sequences used for purification, probing, or priming. For example,heterologous sequences include transcribed, untranslated sequences thatmay play a role in transcription and mRNA processing, such as ribosomebinding and stability of mRNA. The heterologous sequences mayalternatively comprise additional coding sequences that provideadditional functionalities. Thus, a nucleotide sequence encoding apolypeptide may be fused to a tag sequence, such as a sequence encodinga peptide that facilitates purification or detection of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the tag amino acid sequence is a hexa-histidine peptide, suchas the tag provided in a pQE vector (QIAGEN), or in any of a number ofadditional, commercially available vectors. For instance, hexa-histidineprovides for the convenient purification of the fusion protein (see,Gentz et al., 1989, Proc Natl Acad Sci USA February; 86(3):821-4, thedisclosure of which is incorporated by reference in its entirety). The“HA” tag is another peptide useful for purification which corresponds toan epitope derived from the influenza hemagglutinin protein (see,Wilson, et al., 1984, Cell July; 37(3):767-78, the disclosure of whichis incorporated by reference in its entirety). As discussed below, othersuch fusion proteins include a GENSET polypeptide fused to Fc at the N-or C-terminus.

Regulatory Sequences of the Invention

As mentioned, the genomic sequence of GENSET genes contain regulatorysequences in the non-coding 5′-flanking region and possibly in thenon-coding 3′-flanking region that border the GENSET polypeptide codingregions containing the exons of these genes. Polynucleotides derivedfrom GENSET polynucleotide 5′ and 3′ regulatory regions are useful inorder to detect the presence of at least a copy of a genomic nucleotidesequence of the GENSET gene or a fragment thereof in a test sample.

Preferred Regulatory Sequences

Polynucleotides carrying the regulatory elements located at the 5′ endand at the 3′ end of GENSET polypeptide coding regions may beadvantageously used to control, e.g., the transcriptional andtranslational activity of a heterologous polynucleotide of interest.

Thus, the present invention also concerns a purified or isolated nucleicacid comprising a polynucleotide which is selected from the groupconsisting of the 5′ and 3′ GENSET polynucleotide regulatory regions,sequences complementary thereto, regulatory active fragments andvariants thereof.

Another object of the invention consists of purified, isolated orrecombinant nucleic acids comprising a polynucleotide that hybridizes,under the stringent hybridization conditions defined herein, with apolynucleotide of the present invention.

Preferred fragments of 5′ and 3′ regulatory regions are any one integerbetween 20 and 20,000 nucleotides in length.

For the purpose of the invention, a nucleic acid or polynucleotide is“functional” as a “regulatory region” for expressing a recombinantpolypeptide or a recombinant polynucleotide if said regulatorypolynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide. The regulatorypolynucleotides of the invention may be prepared using methods known inthe art.

The regulatory polynucleotides according to the invention may be part ofa recombinant expression vector that may be used to express a codingsequence in a desired host cell or host organism.

Preferred 5′-regulatory polynucleotides of the invention include 5′-UTRsof GENSET cDNAs, or regulatory active fragments or variants thereof.

Preferred 3′-regulatory polynucleotide of the invention include 3′-UTRsof GENSET cDNAs, or regulatory active fragments or variants thereof.

A further object of the invention consists of a purified or isolatednucleic acid comprising:

-   -   a) polynucleotide comprising a 5′ regulatory nucleotide sequence        selected from the group consisting of:        -   (i) a nucleotide sequence comprising a polynucleotide of a            GENSET polynucleotide 5′ regulatory region or a            complementary sequence thereto;        -   (ii) a nucleotide sequence comprising a polynucleotide            having at least 95% of nucleotide identity with the            nucleotide sequence of a GENSET polynucleotide 5′ regulatory            region or a complementary sequence thereto;        -   (iii) a nucleotide sequence comprising a polynucleotide that            hybridizes under stringent hybridization conditions with the            nucleotide sequence of a GENSET polynucleotide 5′ regulatory            region or a complementary sequence thereto; and        -   (iv) a regulatory active fragment or variant of the            polynucleotides in (i), (ii) and (iii);    -   b) a nucleic acid molecule encoding a desired polypeptide or a        nucleic acid molecule of interest, wherein said nucleic acid        molecule is operably linked to the polynucleotide defined in        (a); and    -   c) optionally, a polynucleotide comprising a 3′-regulatory        polynucleotide, preferably a 3′-regulatory polynucleotide of a        GENSET gene.

In a specific embodiment, the nucleic acid defined above includes the5′-UTR of a GENSET cDNA, or a regulatory active fragment or variantthereof.

The regulatory polynucleotide of the 3′ regulatory region, or itsregulatory active fragments or variants, is advantageously operablylinked at the 3′-end of the nucleic acid molecule encoding the desiredpolypeptide or nucleic acid molecule of interest.

The desired polypeptide encoded by the above-described nucleic acid maybe of various nature or origin, encompassing proteins of prokaryoticviral or eukaryotic origin. Among the polypeptides expressed under thecontrol of a GENSET polynucleotide regulatory region include bacterial,fungal or viral antigens. Also encompassed are eukaryotic proteins suchas intracellular proteins, such as “house keeping” proteins,membrane-bound proteins, such as mitochondrial membrane-bound proteinsand cell surface receptors, and secreted proteins such as endogenousmediators such as cytokines. The desired polypeptide may be aheterologous polypeptide or a GENSET polypeptide, especially a proteinwith an amino acid sequence selected from the group consisting of thepolypeptide sequences of the Sequence Listing, those encoded by the cDNAinserts of the deposited clone pool, fragments and variants thereof.

The desired nucleic acids encoded by the above-describedpolynucleotides, usually an RNA molecule, may be complementary to adesired coding polynucleotide, for example to a GENSET coding sequence,and thus useful as an antisense polynucleotide. Such a polynucleotidemay be included in a recombinant expression vector in order to expressthe desired polypeptide or the desired nucleic acid in host cell or in ahost organism. Suitable recombinant vectors that contain apolynucleotide such as described herein are disclosed elsewhere in thespecification.

Polynucleotide Variants

The invention also relates to variants of the polynucleotides describedherein and fragments thereof. “Variants” of polynucleotides, as the termis used herein, are polynucleotides that differ from a referencepolynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical. The present inventionencompasses both allelic variants and degenerate variants.

Allelic Variant

A variant of a polynucleotide may be a naturally occurring variant suchas a naturally occurring allelic variant, or it may be a variant that isnot known to occur naturally. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism [see Lewin, (1989), Proc. Natl. Acad. Sci. USA86:9832-8935], the disclosure of which is incorporated by reference inits entirety. Diploid organisms may be homozygous or heterozygous for anallelic form. Non-naturally occurring variants of the polynucleotide maybe made by art-known mutagenesis techniques, including those applied topolynucleotides, cells or organisms. See, for example, Table III, whichprovides sets of related cDNAs of the invention, e.g. sets of sequencesrepresenting allelic variants of a single gene.

Degenerate Variant

In addition to the isolated polynucleotides of the present invention,and fragments thereof, the invention further includes polynucleotideswhich comprise a sequence substantially different from those describedabove but which, due to the degeneracy of the genetic code, still encodea GENSET polypeptide of the present invention. These polynucleotidevariants are referred to as “degenerate variants” throughout the instantapplication. That is, all possible polynucleotide sequences that encodethe GENSET polypeptides of the present invention are contemplated. Thisincludes the genetic code and species-specific codon preferences knownin the art.

Nucleotide changes present in a variant polynucleotide may be silent,which means that they do not alter the amino acids encoded by thepolynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. In the context of thepresent invention, preferred embodiments are those in which thepolynucleotide variants encode polypeptides which retain substantiallythe same biological properties or activities as the GENSET protein. Morepreferred polynucleotide variants are those containing conservativesubstitutions.

Similar Polynucleotides

Other embodiments of the present invention provide a purified, isolatedor recombinant polynucleotide which is at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to polynucleotide of the present invention.The above polynucleotides are included regardless of whether they encodea polypeptide having a GENSET biological activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving activity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or primer.Uses of the nucleic acid molecules of the present invention that do notencode a polypeptide having GENSET activity include, inter alia,isolating a GENSET gene or allelic variants thereof from a DNA library,and detecting GENSET mRNA expression in biological samples suspected ofcontaining GENSET mRNA or DNA, e.g., by Northern Blot or PCR analysis.

The present invention is further directed to polynucleotides havingsequences at least 50%. 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identity to a polynucleotide, where said polynucleotides do, in fact,encode a polypeptide having a GENSET biological activity. Of course, dueto the degeneracy of the genetic code, one of ordinary skill in the artwill immediately recognize that a large number of the polynucleotides atleast 50%. 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to apolynucleotide selected from the group consisting of polynucleotidesequences of the Sequence Listing and those of human cDNA clone insertsof the deposited clone pool will encode a polypeptide having biologicalactivity. By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the GENSETpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted, inserted, or substituted with another nucleotide. The querysequence may be any polynucleotide of the present invention.

Hybridizing Polynucleotides

In another aspect, the invention provides an isolated or purifiednucleic acid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to any polynucleotide of the present.Such hybridizing polynucleotides may be of at least any one integerbetween 10 and 10,000 nucleotides in length.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a 5′ complementary stretch of T (or U) residues, wouldnot be included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly(A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

Complementary Polynucleotides

The invention further provides isolated nucleic acid molecules having anucleotide sequence fully complementary to any polynucleotide of theinvention.

Polynucleotide Fragments

The present invention is further directed to portions or fragments ofthe polynucleotides of the present invention. Uses for thepolynucleotide fragments of the present invention include probes,primers, molecular weight markers and for expressing the polypeptidefragments of the present invention. Fragments include portions ofpolynucleotides selected from the group consisting of a) polynucleotidesequences of the Sequence Listing, b) genomic GENSET sequences, c)polynucleotides encoding a polypeptide of the present invention, d)sequences of human cDNA clone inserts of the deposited clone pool, ande) polynucleotides encoding the polypeptides encoded by the human cDNAclone inserts of the deposited clone pool. Particularly included in thepresent invention is a purified or isolated polynucleotide comprising atleast 8 consecutive bases of a polynucleotide of the present invention.In one aspect of this embodiment, the polynucleotide comprises at least10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300,400,500, 800, 1000, 1500, or 2000 consecutive nucleotides of apolynucleotide of the present invention.

In addition to the above preferred polynucleotide sizes, furtherpreferred sub-genuses of polynucleotides comprise at least Xnucleotides, wherein “X” is defined as any integer between 8 and theinteger representing the 3′ most nucleotide position as set forth in thesequence listing or elsewhere herein. Further included as preferredpolynucleotides of the present invention are polynucleotide fragments atleast X nucleotides in length, as described above, that are furtherspecified in terms of their 5′ and 3′ position. The 5′ and 3′ positionsare represented by the position numbers set forth in the appendedsequence listing wherein the 5′ most nucleotide is 1 and the 3′ mostnucleotide is the last nucleotide for a particular SEQ ID No. Forallelic, degenerate and other variants, position I is defined as the 5′most nucleotide of the ORF, i.e., the nucleotide “A” of the start codonwith the remaining nucleotides numbered consecutively. Therefore, everycombination of a 5′ and 3′ nucleotide position that a polynucleotidefragment of the present invention, at least 8 contiguous nucleotides inlength, could occupy on a polynucleotide of the invention is included inthe invention as an individual species. The polynucleotide fragmentsspecified by 5′ and 3′ positions can be immediately envisaged and aretherefore not individually listed solely for the purpose of notunnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “a tob”; where “a” equals the 5′ most nucleotide position and “b” equals the3′ most nucleotide position of the polynucleotide; and further where “a”equals an integer between 1 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 8, and where “b”equals an integer between 9 and the number of nucleotides of thepolynucleotide sequence of the present invention; and where “a” is aninteger smaller then “b” by at least 8.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or sub-genuses of polynucleotides specified by size innucleotides as described above. Any number of fragments specified by 5′and 3′ positions or by size in nucleotides, as described above, may beexcluded. Preferred excluded fragments include those having substantialhomology to repeated sequences including Alu, L1, THE and MER repeats,SSTR sequences or satellite, micro-satellite, and telomeric repeats.

Other preferred fragments of the invention are polynucleotidescomprising polynucleotide sequences encoding domains of polypeptides.Such fragments may be used to obtain other polynucleotides encodingpolypeptides having similar domains using hybridization or RT-PCRtechniques. Alternatively, these fragments may be used to express apolypeptide domain which may have a specific biological property.

Another object of the invention is an isolated, purified or recombinantpolynucleotide encoding a polypeptide consisting of, consistingessentially of, or comprising a contiguous span of at least (any integerbetween 5 and 1,000 consecutive amino acids in length more preferably atleast) 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or200 consecutive amino. The present invention further encompasses anycombination of the polynucleotide fragments listed in this section.

Oligonucleotide Primers and Probes

The present invention also encompasses fragments of GENSETpolynucleotides for use as primers and probes. Polynucleotides derivedfrom the GENSET genomic and cDNA sequences are useful in order to detectthe presence of at least a copy of a GENSET polynucleotide or fragment,complement, or variant thereof in a test sample.

Structural Definition

Any polynucleotide of the invention may be used as a primer or probe.Particularly preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of a polynucleotide ofthe present invention.

For amplification purposes, pairs of primers with approximately the sameTm are preferable. Primers may be designed using methods known in theart. Amplification techniques that can be used in the context of thepresent invention include, but are not limited to, the ligase chainreaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A-439 182,the polymerase chain reaction (PCR, RT-PCR) and techniques such as thenucleic acid sequence based amplification (NASBA) described in Guatelliet al., (1990) Proc. Natl. Acad. Sci. USA 35:273-286 and in Compton(1991) Nature 350(6313):91-92, Q-beta amplification as described inEuropean Patent Application No 4544610, strand displacementamplification as described in Walker, et al. (1996), Clin. Chem. 42:9-13and EP A 684 315 and, target mediated amplification as described in PCTPublication WO 9322461, the disclosures of which are incorporated byreference in their entireties.

The probes of the present invention are useful for a number of purposes.They can notably be used in Southern hybridization to genomic DNA. Theprobes can also be used to detect PCR amplification products. They mayalso be used to detect mismatches in the GENSET gene or mRNA using othertechniques. They may also be used to in situ hybridization. Any of thepolynucleotides, primers and probes of the present invention can beconveniently immobilized on a solid support. The solid support is notcritical and can be selected by one skilled in the art. Thus, latexparticles, microparticles, magnetic beads, non-magnetic beads (includingpolystyrene beads), membranes (including nitrocellulose strips), plastictubes, walls of microtiter wells, glass or silicon chips, sheep (orother suitable animal's) red blood cells and duracytes are all suitableexamples. Suitable methods for immobilizing nucleic acids on solidphases include ionic, hydrophobic, covalent interactions and the like. Asolid support, as used herein, refers to any material which isinsoluble, or can be made insoluble by a subsequent reaction. The solidsupport can be chosen for its intrinsic ability to attract andimmobilize the capture reagent. Alternatively, the solid phase canretain an additional receptor which has the ability to attract andimmobilize the capture reagent. The additional receptor can include acharged substance that is oppositely charged with respect to the capturereagent itself or to a charged substance conjugated to the capturereagent. As yet another alternative, the receptor molecule can be anyspecific binding member which is immobilized upon (attached to) thesolid support and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid supportmaterial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, duracytes® and otherconfigurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the invention to a single solidsupport. In addition, polynucleotides other than those of the inventionmay be attached to the same solid support as one or more polynucleotidesof the invention.

Oligonucleotide Array

A substrate comprising a plurality of oligonucleotide primers or probesof the invention may be used either for detecting or amplifying targetedsequences in GENSET genes, may be used for detecting mutations in thecoding or in the non-coding sequences of GENSET genes, and may also beused to determine GENSET gene expression in different contexts such asin different tissues, at different stages of a process (embryodevelopment, disease treatment), and in patients versus healthyindividuals as described elsewhere in the application.

As used herein, the term “array” means a one dimensional, twodimensional, or multidimensional arrangement of nucleic acids ofsufficient length to permit specific detection of gene expression. Forexample, the array may contain a plurality of nucleic acids derived fromgenes whose expression levels are to be assessed. The array may includea GENSET genomic DNA, a GENSET cDNA, sequences complementary thereto orfragments thereof Preferably, the fragments are at least 12, 15, 18, 20,25, 30, 35, 40 or 50 nucleotides in length. More preferably, thefragments are at least 100 nucleotides in length. Even more preferably,the fragments are more than 100 nucleotides in length. In someembodiments the fragments may be more than 500 nucleotides in length.

Any polynucleotide provided herein may be attached in overlapping areasor at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092, which disclosures are hereby incorporated by reference intheir entireties. These arrays may generally be produced using methodsknown in the art, e.g., Fodor et al., (1991) Science 251:767-777, whichdisclosure is hereby incorporated by reference in its entirety. Theimmobilization of arrays of oligonucleotides on solid supports has beenrendered possible by the development of a technology generallyidentified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™)in which, typically, probes are immobilized in a high density array on asolid surface of a chip. Examples of VLSIPS™ technologies are providedin U.S. Pat. Nos. 5,143,854; and 5,412,087 and in PCT Publications WO90/15070, WO 92/10092 and WO 95/11995, which disclosures are herebyincorporated by reference in their entireties. In designing strategiesaimed at providing arrays of nucleotides immobilized on solid supports,further presentation strategies known in the art may be used, such asthose disclosed in PCT Publications WO 94/12305, WO 94/11530, WO97/29212 and WO 97/31256, the disclosures of which are incorporatedherein by reference in their entireties.

Consequently, the invention concerns an array of nucleic acid moleculescomprising at least one polynucleotide of the invention. Preferably, theinvention concerns an array of nucleic acids comprising at least twopolynucleotides of the invention, particularly probes or primers asdescribed herein. Preferably, the invention concerns an array of nucleicacids comprising at least five polynucleotides of the invention,particularly probes or primers as described herein.

Methods of Making the Polynucleotides of the Invention

The present invention also comprises methods of making thepolynucleotides of the invention. Polynucleotides of the invention maybe synthesized either enzymatically using techniques well known to thoseskilled in the art including amplification or hybridization-basedmethods as described herein, or chemically.

A variety of chemical methods of synthesizing nucleic acids are known tothose skilled in the art. In many of these methods, synthesis isconducted on a solid support. Alternatively, polynucleotides may beprepared as described in U.S. Pat. No. 5,049,656, which disclosure ishereby incorporated by reference in its entirety. In some embodiments,several polynucleotides prepared as described above are ligated togetherto generate longer polynucleotides having a desired sequence.

Polypeptides of the Invention

The term “GENSET polypeptides” is used herein to embrace all of theproteins and polypeptides of the present invention. The presentinvention encompasses GENSET polypeptides, including recombinant,isolated or purified GENSET polypeptides consisting of: (a) the fulllength polypeptides of even SEQ ID NOs:2-112; (b) the full lengthpolypeptides encoded by the clone inserts of the deposited clone pool;(c) the epitope-bearing fragments of the polypeptides of even SEQ IDNOs:2-112; (d) the epitope-bearing fragments of the polypeptides encodedby the clone inserts contained in the deposited clone pool; (e) thedomains of the polypeptides of even SEQ ID NOs:2-112; (f) the domains ofthe polypeptides encoded by the clone inserts contained in the depositedclone pool; (g) the signal peptides of the polypeptides of even SEQ IDNOs:2-112 or encoded by the human cDNAs of the deposited clone pool; (h)the mature polypeptides of even SEQ ID Nos:2-112 or encoded by the humancDNAs of the deposited clone pool; and (i) the allelic variantpolypeptides of any of the polypeptides of (a)-(f). Other objects of theinvention are polypeptides encoded by the polynucleotides of theinvention as well as fusion polypeptides comprising such polypeptides.

Polypeptide Variants

The present invention further provides for GENSET polypeptides encodedby allelic and splice variants, orthologs, and/or species homologues.Procedures known in the art can be used to obtain, allelic variants,splice variants, orthologs, and/or species homologues of polynucleotidesencoding polypeptides of the Sequence Listing and polypeptides encodedby the clone inserts of the deposited clone pool, using information fromthe sequences disclosed herein or the clones deposited with the ATCC.

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 50% identical, more preferably atleast 60% identical, and still more preferably 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to a polypeptide of the present invention. Bya polypeptide having an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence of the present invention, itis intended that the amino acid sequence of the subject polypeptide isidentical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. By a polypeptide having an amino acid sequence atleast, for example, 95% “similar” to a query amino acid sequence of thepresent invention, it is intended that the amino acid sequence of thesubject polypeptide is similar (i.e. contains identical or equivalentamino acid residues) to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%similar to a query amino acid sequence, up to 5% (5 of 100) of the aminoacid residues in the subject sequence may be inserted, deleted, (indels)or substituted with another non-equivalent amino acid.

These alterations of the reference sequence may occur at the amino orcarboxy terminal positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. The query sequence maybe an entire amino acid sequence selected from the group consisting ofpolypeptide sequences of the Sequence Listing and those encoded by theclone inserts of the deposited clone pool or any fragment specified asdescribed herein.

The variant polypeptides described herein are included in the presentinvention regardless of whether they have their normal biologicalactivity. This is because even where a particular polypeptide moleculedoes not have biological activity, one of skill in the art would stillknow how to use the polypeptide, for instance, as a vaccine or togenerate antibodies. Other uses of the polypeptides of the presentinvention that do not have GENSET biological activity include, interalia, as epitope tags, in epitope mapping, and as molecular weightmarkers on SDS-PAGE gels or on molecular sieve gel filtration columnsusing methods known to those of skill in the art. As described below,the polypeptides of the present invention can also be used to raisepolyclonal and monoclonal antibodies, which are useful in assays fordetecting GENSET protein expression or as agonists and antagonistscapable of enhancing or inhibiting GENSET protein function. Further,such polypeptides can be used in the yeast two-hybrid system to“capture” GENSET protein binding proteins, which are also candidateagonists and antagonists according to the present invention (see, e.g.,Fields and Song, (1989), Nature, 340: 245-246, which disclosure ishereby incorporated by reference in its entirety).

Preparation of the Polypeptides of the Invention

The polypeptides of the present invention can be prepared in anysuitable manner known in the art. Such polypeptides include isolatednaturally occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. The polypeptides of the present inventionare preferably provided in an isolated form, and may be partially orpreferably substantially purified. Consequently, the present inventionalso comprises methods of making the polypeptides of the invention.

Isolation

From Natural Sources

The GENSET proteins of the invention may be isolated from naturalsources, including bodily fluids, tissues and cells, whether directlyisolated or cultured cells, of humans or non-human animals. Methods forextracting and purifying natural proteins are known in the art, andinclude the use of detergents or chaotropic agents to disrupt particlesfollowed by differential extraction and separation of the polypeptidesby ion exchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis. See, for example,“Methods in Enzymology, Academic Press, 1993” for a variety of methodsfor purifying proteins, which disclosure is hereby incorporated byreference in its entirety. Polypeptides of the invention also can bepurified from natural sources using antibodies directed against thepolypeptides of the invention, such as those described herein, inmethods which are well known in the art of protein purification.

From Recombinant Sources

Preferably, the GENSET polypeptides of the invention are recombinantlyproduced using routine expression methods known in the art. Thepolynucleotide encoding the desired polypeptide is operably linked to apromoter into an expression vector suitable for any convenient host.Both eukaryotic and prokaryotic host systems are used in formingrecombinant polypeptides. The polypeptide is then isolated from lysedcells or from the culture medium and purified to the extent needed forits intended use.

Any polynucleotide of the present invention may be used to expressGENSET polypeptides. The nucleic acid encoding the GENSET polypeptide tobe expressed is operably linked to a promoter in an expression vectorusing conventional cloning technology. The GENSET insert in theexpression vector may comprise the full coding sequence for the GENSETprotein or a portion thereof.

Consequently, a further embodiment of the present invention is a methodof making a polypeptide of the present invention, said method comprisingthe steps of:

-   -   a) obtaining a cDNA comprising a sequence selected from the        group consisting of:        -   i) the polynucleotide sequences of the Sequence Listing,        -   ii) the sequences of human cDNA clone inserts of the            deposited clone pool,        -   iii) polynucleotide sequences encoding one of the            polypeptides of the Sequence Listing, and        -   iv) sequences of polynucleotides encoding a polypeptide            which is encoded by one of the clone insert of the deposited            clone pool;    -   b) inserting said cDNA in an expression vector such that the        cDNA is operably linked to a promoter; and    -   c) introducing said expression vector into a host cell whereby        said host cell produces said polypeptide.

In one aspect of this embodiment, the method further comprises the stepof isolating the polypeptide. Another embodiment of the presentinvention is a polypeptide obtainable by the method described in thepreceding paragraph.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art. Commercially availablevectors and expression systems are available from a variety of suppliersincluding Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla,Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). Ifdesired, to enhance expression and facilitate proper protein folding,the codon context and codon pairing of the sequence is optimized for theparticular expression organism in which the expression vector isintroduced, as explained in U.S. Pat. No. 5,082,767, which disclosure ishereby incorporated by reference in its entirety.

In one embodiment, the entire coding sequence of a GENSET cDNA and the3′ UTR through the poly A signal of the cDNA is operably linked to apromoter in the expression vector. Alternatively, if the nucleic acidencoding a portion of the GENSET protein lacks a methionine to serve asthe initiation site, an initiating methionine can be introduced next tothe first codon of the nucleic acid using conventional techniques.Similarly, if the insert from the GENSET cDNA lacks a poly A signal,this sequence can be added to the construct by, for example, splicingout the Poly A signal from pSG5 (Stratagene) using BglI and SalIrestriction endonuclease enzymes and incorporating it into the mammalianexpression vector pXT1 (Stratagene). pXT1 contains the LTRs and aportion of the gag gene from Moloney Murine Leukemia Virus. The positionof the LTRs in the construct allows efficient stable transfection. Thevector includes the Herpes Simplex Thymidine Kinase promoter and theselectable neomycin gene.

In another embodiment, it is often advantageous to add to therecombinant polynucleotide additional nucleotide sequence which codesfor secretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Transfection of a GENSET expression vector into mouse NTH 3T3 cells isbut one embodiment of introducing polynucleotides into host cells.Introduction of a polynucleotide encoding a polypeptide into a host cellcan be effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, or other methods. Such methods are described inmany standard laboratory manuals, such as Davis et al., (1986) BasicMethods in Molecular Biology, ed., Elsevier Press, NY, which disclosureis hereby incorporated by reference in its entirety. It is specificallycontemplated that the polypeptides of the present invention may in factbe expressed by a host cell lacking a recombinant vector or naturallyproduced by a cell.

Alternatively, the GENSET polypeptide to be expressed may also be aproduct of transgenic animals, i.e., as a component of the milk oftransgenic cows, goats, pigs or sheep which are characterized by somaticor germ cells containing a nucleotide sequence encoding the protein ofinterest.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including differentialextraction, ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. See, for example, “Methods in Enzymology”, supra for avariety of methods for purifying proteins. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.A recombinantly produced version of a GENSET polypeptide can besubstantially purified using techniques described herein or otherwiseknown in the art, such as, for example, by the one-step method describedin Smith and Johnson (1988) Gene. 67(1):31-40, which disclosure ishereby incorporated by reference in its entirety. Polypeptides of theinvention also can be purified from recombinant sources using antibodiesdirected against the polypeptides of the invention, such as thosedescribed herein, in methods which are well known in the art of proteinpurification.

Preferably, the recombinantly expressed GENSET polypeptide is purifiedusing standard immunochromatography techniques such as the one describedin the section entitled “Immunoaffinity Chromatography”. In suchprocedures, a solution containing the protein of interest, such as theculture medium or a cell extract, is applied to a column havingantibodies against the protein attached to the chromatography matrix.The recombinant protein is allowed to bind the immunochromatographycolumn. Thereafter, the column is washed to remove non-specificallybound proteins. The specifically bound secreted protein is then releasedfrom the column and recovered using standard techniques.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked. Thus, specifically included as an aspect of theinvention are polypeptides of the present invention lacking the aminoterminal methionine.

From Chemical Synthesis

In addition, polypeptides of the invention, especially short proteinfragments, can be chemically synthesized using techniques known in theart [See, e.g. Creighton (1983), Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co. 2nd Ed., T. E., New York; and Hunkapilleret al., (1984) Nature. 310(5973):105-11], which disclosures are herebyincorporated by reference in their entireties. For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Alternatively, the methods described in U.S. Pat. No. 5,049,656, whichdisclosure is hereby incorporated by reference in its entirety, may beused.

Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

Modifications

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-inkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity. See U.S. Pat. No.4,179,337. The chemical moieties for derivatization may be selected.See, U.S. Pat. No. 4,179,337 which disclosure is hereby incorporated byreference in its entirety. The chemical moieties for derivatization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,(coupling PEG to G-CSF), and Malik et al., (1992), Exp. Hematol.20:1028-1035 (reporting pegylation of GM-CSF using tresyl chloride),which disclosures are hereby incorporated by reference in theirentireties. For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulffiydryl groups may also be used as a reactive groupfor attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation, which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

Multimerization

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions containing them.In specific embodiments, the polypeptides of the invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term “homomer”, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequences of the SequenceListing or encoded by the human cDNA clone inserts of the depositedclone pool (including fragments, variants, splice variants, and fusionproteins, corresponding to these polypeptides as described herein).These homomers may contain polypeptides having identical or differentamino acid sequences. In a specific embodiment, a homomer of theinvention is a multimer containing only polypeptides having an identicalamino acid sequence. In another specific embodiment, a homomer of theinvention is a multimer containing polypeptides having different aminoacid sequences. In specific embodiments, the multimer of the inventionis a homodimer (e.g., containing polypeptides having identical ordifferent amino acid sequences) or a homotrimer (e.g., containingpolypeptides having identical and/or different amino acid sequences). Inadditional embodiments, the homomenc multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term “heteromer” refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences,which interact in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925, which disclosure is hereby incorporated by referencein its entirety). In a specific example, the covalent associations arebetween the heterologous sequence contained in an Fc fusion protein ofthe invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication No: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves the use of polypeptides of the invention fused to a leucinezipper or isoleucine zipper polypeptide sequence. Leucine zipper andisoleucine zipper domains are polypeptides that promote multimerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins, and have since been found ina variety of different proteins [Landschulz et al., (1988), Science.240:1759]. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al., (1994), FEBS Letters. 344:191 andin U.S. patent application Ser. No. 08/446,922, which disclosure ishereby incorporated by reference in its entirety. Other peptides derivedfrom naturally occurring trimeric proteins may be employed in preparingtrimeric polypeptides of the invention. In another example, proteins ofthe invention are associated by interactions between Flag® polypeptidesequence contained in fusion proteins of the invention containing Flag®polypeptide sequence. In a further embodiment, associations proteins ofthe invention are associated by interactions between heterologouspolypeptide sequence contained in Flag® fusion proteins of the inventionand anti Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, othertechniques known in the art may be applied to generate liposomescontaining the polypeptide components desired to be contained in themultimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Mutated Polypeptides

To improve or alter the characteristics of GENSET polypeptides of thepresent invention, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions, or fusion proteins. Such modifiedpolypeptides can show, e.g., increased/decreased biological activity orincreased/decreased stability. In addition, they may be purified inhigher yields and show better solubility than the corresponding naturalpolypeptide, at least under certain purification and storage conditions.Further, the polypeptides of the present invention may be produced asmultimers including dimers, trimers and tetramers. Multimerization maybe facilitated by linkers or recombinantly though heterologouspolypeptides such as Fc regions.

N- and C-Terminal Deletions

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. [See, e.g., Ron et al., (1993), Biol Chem., 268 2984-2988.]Accordingly, the present invention provides polypeptides having one ormore residues deleted from the amino terminus. Similarly, many examplesof biologically functional C-terminal deletion mutants are known (see,e.g., Dobeli, et al. 1988). Accordingly, the present invention providespolypeptides having one or more residues deleted from the carboxyterminus. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini asdescribed below.

Other Mutations

Other mutants in addition to N- and C-terminal deletion forms of theprotein discussed above are included in the present invention. Thus, theinvention further includes variations of the GENSET polypeptides whichshow substantial GENSET polypeptide activity. Such mutants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as to have little effecton activity.

There are two main approaches for studying the tolerance of an aminoacid sequence to change [see, Bowie et al., (1994), Science.247:1306-1310, which disclosure is hereby incorporated by reference inits entirety]. The first method relies on the process of evolution, inwhich mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. These studies haverevealed that proteins are surprisingly tolerant of amino acidsubstitutions.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Phe;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. Thus, the polypeptide of the presentinvention may be, for example: (i) one in which one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code; or (ii) one in which one or more of the amino acidresidues includes a substituent group; or (iii) one in which the GENSETpolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol); or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the above form of the polypeptide or a pro-proteinsequence.

Thus, the GENSET polypeptides of the present invention may include oneor more amino acid substitutions, deletions, or additions, either fromnatural mutations or human manipulation. As indicated, changes arepreferably of a minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein. The following groups of amino acids represent equivalentchanges: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser,Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe,Tyr, Trp, His.

Furthermore, GENSET polypeptides of the present invention may includeone or more amino acid substitutions that mimic modified amino acids. Anexample of this type of substitution includes replacing amino acids thatare capable of being phosphorylated (e.g., serine, threonine, ortyrosine) with a negatively charged amino acid that resembles thenegative charge of the phosphorylated amino acid (e.g., aspartic acid orglutamic acid). Also included is substitution of amino acids that arecapable of being modified by hydrophobic groups (e.g., arginine) withamino acids carrying bulky hydrophobic side chains, such as tryptophanor phenylalanine. Therefore, a specific embodiment of the inventionincludes GENSET polypeptides that include one or more amino acidsubstitutions that mimic modified amino acids at positions where aminoacids that are capable of being modified are normally positioned.Further included are GENSET polypeptides where any subset of modifiableamino acids are substituted. For example, a GENSET polypeptide thatincludes three serine residues may be substituted at any one, any two,or all three of said serines. Furthermore, any GENSET polypeptide aminoacid capable of being modified may be excluded from substitution with amodification-mimicking amino acid.

A specific embodiment of a modified GENSET peptide molecule of interestaccording to the present invention, includes, but is not limited to, apeptide molecule which is resistant to proteolysis, is a peptide inwhich the —CONH— peptide bond is modified and replaced by a (CH2NH)reduced bond, a (NHCO) retro inverso bond, a (CH2—O) methylene-oxy bond,a (CH2—S) thiomethylene bond, a (CH2CH2) carba bond, a (CO—CH2)cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond), a (N—N) bound, aE-alcene bond or also a —CH═CH— bond. The invention also encompasses ahuman GENSET polypeptide or a fragment or a variant thereof in which atleast one peptide bond has been modified as described above.

Amino acids in the GENSET proteins of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis [see,e.g., Cunningham et al. (1989), Science 244:1081-1085, which disclosureis hereby incorporated by reference in its entirety]. Of specialinterest are substitutions of charged amino acids with other charged orneutral amino acids which may produce proteins with highly desirableimproved characteristics, such as less aggregation. Aggregation may notonly reduce activity but also be problematic when preparingpharmaceutical formulations, because aggregates can be immunogenic.[See, e.g., Pinckard et al., (1967), Clin. Exp. Immunol 2:331-340;Robbins et al, (1987), Diabetes. 36:838-845; and Cleland et al., (1993),Crit. Rev. Therapeutic Drug Carrier Systems. 10:307-377.]

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a GENSET polypeptide having anamino acid sequence which contains at least any one integer from 1 to 50of conservative amino acid substitutions. Further included arepolypeptides that contain not more than 40 conservative amino acidsubstitutions, not more than 30 conservative amino acid substitutions,and not more than 20 conservative amino acid substitutions. Alsoprovided are polypeptides which comprise the amino acid sequence of aGENSET polypeptide, having at least one, but not more than 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 conservative amino acid substitutions. Furtherprovided are conservative amino acid substitutions at any appropriateposition or combination of appropriate positions whereby all possiblespecies are included as embodiments of the present invention. Eachconservative substitution or combination of substitutions may also beexcluded.

Polypeptide Fragments

Structural Definition

The present invention is further directed to fragments of thepolypeptides of the present invention. More specifically, the presentinvention embodies purified, isolated, and recombinant polypeptidescomprising at least any one integer between 6 and 1000 (or the length ofthe polypeptides amino acid residues minus 1 if the length is less than1000) of consecutive amino acid residues. Preferably, the fragments areat least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25,30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300consecutive amino acids of a polypeptide of the present invention.

In addition to the above polypeptide fragments, further preferredsub-genuses of polypeptides comprise at least X amino acids, wherein “X”is defined as any integer between 6 and the integer representing theC-terminal amino acid of the polypeptide of the present inventionincluding the polypeptide sequences of the sequence listing below.Further included are species of polypeptide fragments at least 6 aminoacids in length, as described above, that are further specified in termsof their N-terminal and C-terminal positions. However, included in thepresent invention as individual species are all polypeptide fragments,at least 6 amino acids in length, as described above, and may beparticularly specified by a N-terminal and C-terminal position. That is,every combination of a N-terminal and C-terminal position that afragment at least 6 contiguous amino acid residues in length couldoccupy, on any given amino acid sequence of the sequence listing or ofthe present invention is included in the present invention

Further preferred polypeptide fragments comprising amino acids of thesequences of the EVEN numbered SEQ ID NOs. of the Sequence listing, andpolynucleotides encoding the same, are selected from the groupconsisting of amino acids consecutively numbered from 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32,1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44,1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56,1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68,1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80,1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92,1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, 1-103,1-104, 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113,1-114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122, 1-123,1-124, 1-125, 1-126, 1-127, 1-128, 1-129, 1-130, 1-131, 1-132, 1-133,1-134, 1-135, 1-136, 1-137, 1-138, 1-139, 1-140, 1-141, 1-142, 1-143,1-144, 1-145, 1-146, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1-153,1-154, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160, 1-161, 1-162, 1-163,1-164, 1-165, 1-166, 1-167, 1-168, 1-169, 1-170, 1-171, 1-172, 1-173,1-174, 1-175, 1-176, 1-177, 1-178, 1-179, 1-180, 1-181, 1-182, 1-183,1-184, 1-185, 1-186, 1-187, 1-188, 1-189, 1-190, 1-191, 1-192, 1-193,1-194, 1-195, 1-196, 1-197, 1-198, 1-199, 1-200, 1-201, 1-202, 1-203,1-204, 1-205, 1-206, 1-207, 1-208, 1-209, 1-210, 1-211, 1-212, 1-213,1-214, 1-215, 1-216, 1-217, 1-218, 1-219, 1-220, 1-221, 1-222, 1-223,1-224, 1-225, 1-226, 1-227, 1-228, 1-229, 1-230, 1-231, 1-232, 1-233,1-234, 1-235, 1-236, 1-237, 1-238, 1-239, 1-240, 1-241, 1-242, 1-243,1-244, 1-245, 1-246, 1-247, 1-248, 1-249, 1-250, 1-251, 1-252, 1-253,1-254, 1-255, 1-256, 1-257, 1-258, 1-259, 1-260, 1-261, 1-262, 1-263,1-264, 1-265, 1-266, 1-267, 1-268, 1-269, 1-270, 1-271, 1-272, 1-273,1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283,1-284, 1-285, 1-286, 1-287, 1-288, 1-289, 1-290, 1-291, 1-292, 1-293,1-294, 1-295, 1-296, 1-297, 1-298, 1-299, 1-300, 1-301, 1-302, 1-303,1-304, 1-305, 1-306, 1-307, 1-308, 1-309, 1-310, 1-311, 1-312, 1-313,1-314, 1-315, 1-316, 1-317, 1-318, 1-319, 1-320, 1-321, 1-322, 1-323,1-324, 1-325, 1-326, 1-327, 1-328, 1-329, 1-330, 1-331, 1-332, 1-333,1-334, 1-335, 1-336, 1-337, 1-338, 1-339, 1-340, 1-341, 1-342, 1-343,1-344, 1-345, 1-346, 1-347, 1-348, 1-349, 1-350, 1-351, 1-352, 1-353,1-354, 1-355, 1-356, 1-357, 1-358, 1-359, 1-360, 1-361, 1-362, 1-363,1-364, 1-365, 1-366, 1-367, 1-368, 1-369, 1-370, 1-371, 1-372, 1-373,1-374, 1-375, 1-376, 1-377, 1-378, 1-379, 1-380, 1-381, 1-382, 1-383,1-384, 1-385, 1-386, 1-387, 1-388, 1-389, 1-390, 1-391, 1-392, 1-393,1-394, 1-395, 1-396, 1-397, 1-398, 1-399, 1-400, 1-401, 1-402, 1-403,1-404, 1-405, 1-406, 1-407, 1-408, 1-409, 1-410, 1-411, 1-412, 1-413,1-414, 1-415, 1-416, 1-417, 1-418, 1-419, 1-420, 1-421, 1-422, 1-423,1-424, 1-425, 1-426, 1-427, 1-428, 1-429, 1-430, 1-431, 1-432, 1-433,1-434, 1-435, 1-436, 1-437, 1-438, 1-439, 1-440, 1-441, 1-442, 1-443,1-444, 1-445, 1-446, 1-447, 1-448, 1-449, 1-450, 1-451, 1-452, 1-453,1-454, 1-455, 1-456, 1-457, 1-458, 1-459, 1-460, 1-461, 1-462, 1-463,1-464, 1-465, 1-466, 1-467, 1-468, 1-469, 1-470, 1-471, 1-472, 1-473,1-474, 1-475, 1-476, 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483,1-484, 1-485, 1-486, 1-487, 1-488, 1-489, 1-490, 1-491, 1-492, 1-493,1-494, 1-495, 1-496, 1-497, 1-498, 1-499, 1-500, 1-501, 1-502, 1-503,1-504, 1-505, 1-506, 1-507, 1-508, 1-509, 1-510, 1-511, 1-512, 1-513,1-514, 1-515, 1-516, 1-517, 1-518, 1-519, 1-520, 1-521, 1-522, 1-523,1-524, 1-525, 1-526, 1-527, 1-528, 1-529, 1-530, 1-531, 1-532, 1-533,1-534, 1-535, 1-536, 1-537, 1-538, 1-539, 1-540, 1-541, 1-542, 1-543,1-544, 1-545, 1-546, 1-547, 1-548, 1-549, 1-550, 1-551, 1-552, 1-553,1-554, 1-555, 1-556, 1-557, 1-558, 1-559, 1-560, 1-561, 1-562, 1-563,1-564, 1-565, 1-566, 1-567, 1-568, 1-569, 1-570, 1-571, 1-572, 1-573,1-574, 1-575, 1-576, 1-577, 1-578, 1-579, 1-580, 1-581, 1-582, 1-583,1-584, 1-585, 1-586, 1-587, 1-588, 1-589, 1-590, 1-591, 1-592, 1-593,1-594, 1-595, 1-596, 1-597, 1-598, 1-599, 1-600, 1-601, 1-602, 1-603,1-604, 1-605, 1-606, 1-607, 1-608, 1-609, 1-610, 1-611, 1-612, 1-613,1-614, 1-615, 1-616, 1-617, 1-618, 1-619, 1-620, 1-621, 1-622, 1-623,1-624, 1-625, 1-626, 1-627, 1-628, 1-629, 1-630, 1-631, 1-632, 1-633,1-634, 1-635, 1-636, 1-637, 1-638, 1-639, 1-640, 1-641, 1-642, 1-643,1-644, 1-645, 1-646, 1-647, 1-648, 1-649, 1-650, 1-651, 1-652, 1-653,1-654, 1-655, 1-656, 1-657, 1-658, 1-659, 1-660, 1-661, 1-662, 1-663,1-664, 1-665, 1-666, 1-667, 1-668, 1-669, 1-670, 1-671, 1-672, 1-673,1-674, 1-675, 1-676, 1-677, 1-678, 1-679, 1-680, 1-681, 1-682, 1-683,1-684, 1-685, 1-686, 1-687, 1-688, 1-689, 1-690, 1-691, 1-692, 1-693,1-694, 1-695, 1-696, 1-697, 1-698, 1-699, 1-700, 1-701, 1-702, 1-703,1-704, 1-705, 1-706, 1-707, 1-708, 1-709, 1-710, 1-711, 1-712, 1-713,1-714, 1-715, 1-716, 1-717, 1-718, 1-719, 1-720, 1-721, 1-722, 1-723,1-724, 1-725, 1-726, 1-727, 1-728, 1-729, 1-730, 1-731, 1-732, 1-733,1-734, 1-735, 1-736, 1-737, 1-738, 1-739, 1-740, 1-741, 1-742, 1-743,1-744, 1-745, 1-746, 1-747, 1-748, 1-749, 1-750, 1-751, 1-752, 1-753,1-754, 1-755, 1-756, 1-757, 1-758, 1-759, 1-760, 1-761, 1-762, 1-763,1-764, 1-765, 1-766, 1-767, 1-768, 1-769, 1-770, 1-771, 1-772, 1-773,1-774, 1-775, 1-776, 1-777, 1-778, 1-779, 1-780, 1-781, 1-782, 1-783,1-784, 1-785, 1-786, 1-787, 2-787, 3-787, 4-787, 5-787, 6-787, 7-787,8-787, 9-787, 10-787, 11-787, 12-787, 13-787, 14-787, 15-787, 16-787,17-787, 18-787, 19-787, 20-787, 21-787, 22-787, 23-787, 24-787, 25-787,26-787, 27-787, 28-787, 29-787, 30-787, 31-787, 32-787, 33-787, 34-787,35-787, 36-787, 37-787, 38-787, 39-787, 40-787, 41-787, 42-787, 43-787,44-787, 45-787, 46-787, 47-787, 48-787, 49-787, 50-787, 51-787, 52-787,53-787, 54-787, 55-787, 56-787, 57-787, 58-787, 59-787, 60-787, 61-787,62-787, 63-787, 64-787, 65-787, 66-787, 67-787, 68-787, 69-787, 70-787,71-787, 72-787, 73-787, 74-787, 75-787, 76-787, 77-787, 78-787, 79-787,80-787, 81-787, 82-787, 83-787, 84-787, 85-787, 86-787, 87-787, 88-787,89-787, 90-787, 91-787, 92-787, 93-787, 94-787, 95-787, 96-787, 97-787,98-787, 99-787, 100-787, 101-787, 102-787, 103-787, 104-787, 105-787,106-787, 107-787, 108-787, 109-787, 110-787, 111-787, 112-787, 113-787,114-787, 115-787, 116-787, 117-787, 118-787, 119-787, 120-787, 121-787,122-787, 123-787, 124-787, 125-787, 126-787, 127-787, 128-787, 129-787,130-787, 131-787, 132-787, 133-787, 134-787, 135-787, 136-787, 137-787,138-787, 139-787, 140-787, 141-787, 142-787, 143-787, 144-787, 145-787,146-787, 147-787, 148-787, 149-787, 150-787, 151-787, 152-787, 153-787,154-787, 155-787, 156-787, 157-787, 158-787, 159-787, 160-787, 161-787,162-787, 163-787, 164-787, 165-787, 166-787, 167-787, 168-787, 169-787,170-787, 171-787, 172-787, 173-787, 174-787, 175-787, 176-787, 177-787,178-787, 179-787, 180-787, 181-787, 182-787, 183-787, 184-787, 185-787,186-787, 187-787, 188-787, 189-787, 190-787, 191-787, 192-787, 193-787,194-787, 195-787, 196-787, 197-787, 198-787, 199-787, 200-787, 201-787,202-787, 203-787, 204-787, 205-787, 206-787, 207-787, 208-787, 209-787,210-787, 211-787, 212-787, 213-787, 214-787, 215-787, 216-787, 217-787,218-787, 219-787, 220-787, 221-787, 222-787, 223-787, 224-787, 225-787,226-787, 227-787, 228-787, 229-787, 230-787, 231-787, 232-787, 233-787,234-787, 235-787, 236-787, 237-787, 238-787, 239-787, 240-787, 241-787,242-787, 243-787, 244-787, 245-787, 246-787, 247-787, 248-787, 249-787,250-787, 251-787, 252-787, 253-787, 254-787, 255-787, 256-787, 257-787,258-787, 259-787, 260-787, 261-787, 262-787, 263-787, 264-787, 265-787,266-787, 267-787, 268-787, 269-787, 270-787, 271-787, 272-787, 273-787,274-787, 275-787, 276-787, 277-787, 278-787, 279-787, 280-787, 281-787,282-787, 283-787, 284-787, 285-787, 286-787, 287-787, 288-787, 289-787,290-787, 291-787, 292-787, 293-787, 294-787, 295-787, 296-787, 297-787,298-787, 299-787, 300-787, 301-787, 302-787, 303-787, 304-787, 305-787,306-787, 307-787, 308-787, 309-787, 310-787, 311-787, 312-787, 313-787,314-787, 315-787, 316-787, 317-787, 318-787, 319-787, 320-787, 321-787,322-787, 323-787, 324-787, 325-787, 326-787, 327-787, 328-787, 329-787,330-787, 331-787, 332-787, 333-787, 334-787, 335-787, 336-787, 337-787,338-787, 339-787, 340-787, 341-787, 342-787, 343-787, 344-787, 345-787,346-787, 347-787, 348-787, 349-787, 350-787, 351-787, 352-787, 353-787,354-787, 355-787, 356-787, 357-787, 358-787, 359-787, 360-787, 361-787,362-787, 363-787, 364-787, 365-787, 366-787, 367-787, 368-787, 369-787,370-787, 371-787, 372-787, 373-787, 374-787, 375-787, 376-787, 377-787,378-787, 379-787, 380-787, 381-787, 382-787, 383-787, 384-787, 385-787,386-787, 387-787, 388-787, 389-787, 390-787, 391-787, 392-787, 393-787,394-787, 395-787, 396-787, 397-787, 398-787, 399-787, 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512-787, 513-787,514-787, 515-787, 516-787, 517-787, 518-787, 519-787, 520-787, 521-787,522-787, 523-787, 524-787, 525-787, 526-787, 527-787, 528-787, 529-787,530-787, 531-787, 532-787, 533-787, 534-787, 535-787, 536-787, 537-787,538-787, 539-787, 540-787, 541-787, 542-787, 543-787, 544-787, 545-787,546-787, 547-787, 548-787, 549-787, 550-787, 551-787, 552-787, 553-787,554-787, 555-787, 556-787, 557-787, 558-787, 559-787, 560-787, 561-787,562-787, 563-787, 564-787, 565-787, 566-787, 567-787, 568-787, 569-787,570-787, 571-787, 572-787, 573-787, 574-787, 575-787, 576-787, 577-787,578-787, 579-787, 580-787, 581-787, 582-787, 583-787, 584-787, 585-787,586-787, 587-787, 588-787, 589-787, 590-787, 591-787, 592-787, 593-787,594-787, 595-787, 596-787, 597-787, 598-787, 599-787, 600-787, 601-787,602-787, 603-787, 604-787, 605-787, 606-787, 607-787, 608-787, 609-787,610-787, 611-787, 612-787, 613-787, 614-787, 615-787, 616-787, 617-787,618-787, 619-787, 620-787, 621-787, 622-787, 623-787, 624-787, 625-787,626-787, 627-787, 628-787, 629-787, 630-787, 631-787, 632-787, 633-787,634-787, 635-787, 636-787, 637-787, 638-787, 639-787, 640-787, 641-787,642-787, 643-787, 644-787, 645-787, 646-787, 647-787, 648-787, 649-787,650-787, 651-787, 652-787, 653-787, 654-787, 655-787, 656-787, 657-787,658-787, 659-787, 660-787, 661-787, 662-787, 663-787, 664-787, 665-787,666-787, 667-787, 668-787, 669-787, 670-787, 671-787, 672-787, 673-787,674-787, 675-787, 676-787, 677-787, 678-787, 679-787, 680-787, 681-787,682-787, 683-787, 684-787, 685-787, 686-787, 687-787, 688-787, 689-787,690-787, 691-787, 692-787, 693-787, 694-787, 695-787, 696-787, 697-787,698-787, 699-787, 700-787, 701-787, 702-787, 703-787, 704-787, 705-787,706-787, 707-787, 708-787, 709-787, 710-787, 711-787, 712-787, 713-787,714-787, 715-787, 716-787, 717-787, 718-787, 719-787, 720-787, 721-787,722-787, 723-787, 724-787, 725-787, 726-787, 727-787, 728-787, 729-787,730-787, 731-787, 732-787, 733-787, 734-787, 735-787, 736-787, 737-787,738-787, 739-787, 740-787, 741-787, 742-787, 743-787, 744-787, 745-787,746-787, 747-787, 748-787, 749-787, 750-787, 751-787, 752-787, 753-787,754-787, 755-787, 756-787, 757-787, 758-787, 759-787, 760-787, 761-787,762-787, 763-787, 764-787, 765-787, 766-787, 767-787, 768-787, 769-787,770-787, 771-787, 772-787, 773-787, 774-787, 775-787, 776-787, 777-787,778-787, 779-787, 780-787, 781-787, 782-787, 2-786, 3-785, 4-784, 5-783,6-782, 7-781, 8-780, 9-779, 10-778, 11-777, 12-776, 13-775, 14-774,15-773, 16-772, 17-771, 18-770, 19-769, 20-768, 21-767, 22-766, 23-765,24-764, 25-763, 26-762, 27-761, 28-760, 29-759, 30-758, 31-757, 32-756,33-755, 34-754, 35-753, 36-752, 37-751, 38-750, 39-749, 40-748, 41-747,42-746, 43-745, 44-744, 45-743, 46-742, 47-741, 48-740, 49-739, 50-738,51-737, 52-736, 53-735, 54-734, 55-733, 56-732, 57-731, 58-730, 59-729,60-728, 61-727, 62-726, 63-725, 64-724, 65-723, 66-722, 67-721, 68-720,69-719, 70-718, 71-717, 72-716, 73-715, 74-715, 75-714, 76-712, 77-711,78-710, 79-709, 80-708, 81-707, 82-706, 83-705, 84-704, 85-703, 86-702,87-701, 88-700, 89-699, 90-698, 91-697, 92-696, 93-695, 94-694, 95-693,96-692, 97-691, 98-690, 99-689, 100-688, 101-687, 102-686, 103-685,104-684, 105-683, 106-682, 107-681, 108-680, 109-679, 110-678, 111-677,112-676, 113-675, 114-674, 115-673, 116-672, 117-671, 118-670, 119-669,120-668, 121-667, 122-666, 123-665, 124-664, 125-663, 126-662, 127-661,128-660, 129-659, 130-658, 131-657, 132-656, 133-655, 134-654, 135-653,136-652, 137-651, 138-650, 139-649, 140-648, 141-647, 142-646, 143-645,144-644, 145-643, 146-642, 147-641, 148-640, 149-639, 150-638, 151-637,152-636, 153-635, 154-634, 155-633, 156-632, 157-631, 158-630, 159-629,160-628, 161-627, 162-626, 163-625, 164-624, 165-623, 166-622, 167-621,168-620, 169-619, 170-618, 171-617, 172-616, 173-615, 174-614, 175-613,176-612, 177-611, 178-610, 179-609, 180-608, 181-607, 182-606, 183-605,184-604, 185-603, 186-602, 187-601, 188-600, 189-599, 190-598, 191-597,192-596, 193-595, 194-594, 195-593, 196-592, 197-591, 198-590, 199-589,200-588, 201-587, 202-586, 203-585, 204-584, 205-583, 206-582, 207-581,208-580, 209-579, 210-578, 211-577, 212-576, 213-575, 214-574, 215-573,216-572, 217-571, 218-570, 219-569, 220-568, 221-567, 222-566, 223-565,224-564, 225-563, 226-562, 227-561, 228-560, 229-559, 230-558, 231-557,232-556, 233-555, 234-554, 235-553, 236-552, 237-551, 238-550, 239-549,240-548, 241-547, 242-546, 243-545, 244-544, 245-543, 246-542, 247-541,248-540, 249-539, 250-538, 251-537, 252-536, 253-535, 254-534, 255-533,256-532, 257-531, 258-530, 259-529, 260-528, 261-527, 262-526, 263-525,264-524, 265-523, 266-522, 267-521, 268-520, 269-519, 270-518, 271-517,272-516, 273-515, 274-514, 275-513, 276-512, 277-511, 278-510, 279-509,280-508, 281-507, 282-506, 283-505, 284-504, 285-503, 286-502, 287-501,288-500, 289-499, 290-498, 291-497, 292-496, 293-495, 294-494, 295-493,296-492, 297-491, 298-490, 299-489, 300-488, 301-487, 302-486, 303-485,304-484, 305-483, 306-482, 307-481, 308-480, 309-479, 310-478, 311-477,312-476, 313-475, 314-474, 315-473, 316-472, 317-471, 318-470, 319-469,320-468, 321-467, 322-466, 323-465, 324-464, 325-463, 326-462, 327-461,328-460, 329-459, 330-458, 331-457, 332-456, 333-455, 334-454, 335-453,336-452, 337-451, 338-450, 339-449, 340-448, 341-447, 342-446, 343-445,344-444, 345-443, 346-442, 347-441, 348-440, 349-439, 350-438, 351-437,352-436, 353-435, 354-434, 355-433, 356-432, 357-431, 358-430, 359-429,360-428, 361-427, 362-426, 363-425, 364-424, 365-423, 366-422, 367-421,368-420, 369-419, 370-418, 371-417, 372-416, 373-415, 374-414, 375-413,376-412, 377-411, 378-410, 379-409, 380-408, 381-407, 382-406, 383-405,384-404, 385-403, 386-402, 387-401, 388-400, 389-399, 390-398, and391-397, wherein the numbering of amino acids comprising any onefragment is consistent with the polypeptide sequence of any one EVENnumbered SEQ ID of the Sequence listing.

Further preferred polypeptide fragments of the EVEN numbered SEQ ID NOs.of the Sequence listing, and polynucleotides encoding the same, areselected from the group consisting of fragments comprising any 50consecutive amino acids numbered from 1-50, 2-51, 3-52, 4-53, 5-54,6-55, 7-56, 8-57, 9-58, 10-59, 11-60, 12-61, 13-62, 14-63, 15-64, 16-65,17-66, 18-67, 19-68, 20-69, 21-70, 22-71, 23-72, 24-73, 25-74, 26-75,27-76, 28-77, 29-78, 30-79, 31-80, 32-81, 33-82, 34-83, 35-84, 36-85,37-86, 38-87, 39-88, 40-89, 41-90, 42-91, 43-92, 44-93, 45-94, 46-95,47-96, 48-97, 49-98, 50-99, 51-100, 52-101, 53-102, 54-103, 55-104,56-105, 57-106, 58-107, 59-108, 60-109, 61-110, 62-111, 63-112, 64-113,65-114, 66-115, 67-116, 68-117, 69-118, 70-119, 71-120, 72-121, 73-122,74-123, 75-124, 76-125, 77-126, 78-127, 79-128, 80-129, 81-130, 82-131,83-132, 84-133, 85-134, 86-135, 87-136, 88-137, 89-138, 90-139, 91-140,92-141, 93-142, 94-143, 95-144, 96-145, 97-146, 98-147, 99-148, 100-149,101-150, 102-151, 103-152, 104-153, 105-154, 106-155, 107-156, 108-157,109-158, 110-159, 111-160, 112-161, 113-162, 114-163, 115-164, 116-165,117-166, 118-167, 119-168, 120-169, 121-170, 122-171, 123-172, 124-173,125-174, 126-175, 127-176, 128-177, 129-178, 130-179, 131-180, 132-181,133-182, 134-183, 135-184, 136-185, 137-186, 138-187, 139-188, 140-189,141-190, 142-191, 143-192, 144-193, 145-194, 146-195, 147-196, 148-197,149-198, 150-199, 151-200, 152-201, 153-202, 154-203, 155-204, 156-205,157-206, 158-207, 159-208, 160-209, 161-210, 162-211, 163-212, 164-213,165-214, 166-215, 167-216, 168-217, 169-218, 170-219, 171-220, 172-221,173-222, 174-223, 175-224, 176-225, 177-226, 178-227, 179-228, 180-229,181-230, 182-231, 183-232, 184-233, 185-234, 186-235, 187-236, 188-237,189-238, 190-239, 191-240, 192-241, 193-242, 194-243, 195-244, 196-245,197-246, 198-247, 199-248, 200-249, 201-250, 202-251, 203-252, 204-253,205-254, 206-255, 207-256, 208-257, 209-258, 210-259, 211-260, 212-261,213-262, 214-263, 215-264, 216-265, 217-266, 218-267, 219-268, 220-269,221-270, 222-271, 223-272, 224-273, 225-274, 226-275, 227-276, 228-277,229-278, 230-279, 231-280, 232-281, 233-282, 234-283, 235-284, 236-285,237-286, 238-287, 239-288, 240-289, 241-290, 242-291, 243-292, 244-293,245-294, 246-295, 247-296, 248-297, 249-298, 250-299, 251-300, 252-301,253-302, 254-303, 255-304, 256-305, 257-306, 258-307, 259-308, 260-309,261-310, 262-311, 263-312, 264-313, 265-314, 266-315, 267-316, 268-317,269-318, 270-319, 271-320, 272-321, 273-322, 274-323, 275-324, 276-325,277-326, 278-327, 279-328, 280-329, 281-330, 282-331, 283-332, 284-333,285-334, 286-335, 287-336, 288-337, 289-338, 290-339, 291-340, 292-341,293-342, 294-343, 295-344, 296-345, 297-346, 298-347, 299-348, 300-349,301-350, 302-351, 303-352, 304-353, 305-354, 306-355, 307-356, 308-357,309-358, 310-359, 311-360, 312-361, 313-362, 314-363, 315-364, 316-365,317-366, 318-367, 319-368, 320-369, 321-370, 322-371, 323-372, 324-373,325-374, 326-375, 327-376, 328-377, 329-378, 330-379, 331-380, 332-381,333-382, 334-383, 335-384, 336-385, 337-386, 338-387, 339-388, 340-389,341-390, 342-391, 343-392, 344-393, 345-394, 346-395, 347-396, 348-397,349-398, 350-399, 351-400, 352-401, 353-402, 354-403, 355-404, 356-405,357-406, 358-407, 359-408, 360-409, 361-410, 362-411, 363-412, 364-413,365-414, 366-415, 367-416, 368-417, 369-418, 370-419, 371-420, 372-421,373-422, 374-423, 375-424, 376-425, 377-426, 378-427, 379-428, 380-429,381-430, 382-431, 383-432, 384-433, 385-434, 386-435, 387-436, 388-437,389-438, 390-439, 391-440, 392-441, 393-442, 394-443, 395-444, 396-445,397-446, 398-447, 399-448, 400-449, 401-450, 402-451, 403-452, 404-453,405-454, 406-455, 407-456, 408-457, 409-458, 410-459, 411-460, 412-461,413-462, 414-463, 415-464, 416-465, 417-466, 418-467, 419-468, 420-469,421-470, 422-471, 423-472, 424-473, 425-474, 426-475, 427-476, 428-477,429-478, 430-479, 431-480, 432-481, 433-482, 434-483, 435-484, 436-485,437-486, 438-487, 439-488, 440-489, 441-490, 442-491, 443-492, 444-493,445-494, 446-495, 447-496, 448-497, 449-498, 450-499, 451-500, 452-501,453-502, 454-503, 455-504, 456-505, 457-506, 458-507, 459-508, 460-509,461-510, 462-511, 463-512, 464-513, 465-514, 466-515, 467-516, 468-517,469-518, 470-519, 471-520, 472-521, 473-522, 474-523, 475-524, 476-525,477-526, 478-527, 479-528, 480-529, 481-530, 482-531, 483-532, 484-533,485-534, 486-535, 487-536, 488-537, 489-538, 490-539, 491-540, 492-541,493-542, 494-543, 495-544, 496-545, 497-546, 498-547, 499-548, 500-549,501-550, 502-551, 503-552, 504-553, 505-554, 506-555, 507-556, 508-557,509-558, 510-559, 511-560, 512-561, 513-562, 514-563, 515-564, 516-565,517-566, 518-567, 519-568, 520-569, 521-570, 522-571, 523-572, 524-573,525-574, 526-575, 527-576, 528-577, 529-578, 530-579, 531-580, 532-581,533-582, 534-583, 535-584, 536-585, 537-586, 538-587, 539-588, 540-589,541-590, 542-591, 543-592, 544-593, 545-594, 546-595, 547-596, 548-597,549-598, 550-599, 551-600, 552-601, 553-602, 554-603, 555-604, 556-605,557-606, 558-607, 559-608, 560-609, 561-610, 562-611, 563-612, 564-613,565-614, 566-615, 567-616, 568-617, 569-618, 570-619, 571-620, 572-621,573-622, 574-623, 575-624, 576-625, 577-626, 578-627, 579-628, 580-629,581-630, 582-631, 583-632, 584-633, 585-634, 586-635, 587-636, 588-637,589-638, 590-639, 591-640, 592-641, 593-642, 594-643, 595-644, 596-645,597-646, 598-647, 599-648, 600-649, 601-650, 602-651, 603-652, 604-653,605-654, 606-655, 607-656, 608-657, 609-658, 610-659, 611-660, 612-661,613-662, 614-663, 615-664, 616-665, 617-666, 618-667, 619-668, 620-669,621-670, 622-671, 623-672, 624-673, 625-674, 626-675, 627-676, 628-677,629-678, 630-679, 631-680, 632-681, 633-682, 634-683, 635-684, 636-685,637-686, 638-687, 639-688, 640-689, 641-690, 642-691, 643-692, 644-693,645-694, 646-695, 647-696, 648-697, 649-698, 650-699, 651-700, 652-701,653-702, 654-703, 655-704, 656-705, 657-706, 658-707, 659-708, 660-709,661-710, 662-711, 663-712, 664-713, 665-714, 666-715, 667-716, 668-717,669-718, 670-719, 671-720, 672-721, 673-722, 674-723, 675-724, 676-725,677-726, 678-727, 679-728, 680-729, 681-730, 682-731, 683-732, 684-733,685-734, 686-735, 687-736, 688-737, 689-738, 690-739, 691-740, 692-741,693-742, 694-743, 695-744, 696-745, 697-746, 698-747, 699-748, 700-749,701-750, 702-751, 703-752, 704-753, 705-754, 706-755, 707-756, 708-757,709-758, 710-759, 711-760, 712-761, 713-762, 714-763, 715-764, 716-765,717-766, 718-767, 719-768, 720-769, 721-770, 722-771, 723-772, 724-773,725-774, 726-775, 727-776, 728-777, 729-778,730-779, 731-780, 732-781,733-782, 734-783, 735-784, 736-785, 737-786, and 738-787, wherein thenumbering of amino acids comprising any one fragment is consistent withthe polypeptide sequence of any one EVEN numbered SEQ ID of the Sequencelisting.

Further preferred polypeptide fragments of the EVEN numbered SEQ ID NOs.of the Sequence listing, and polynucleotides encoding the same, areselected from the group consisting of fragments comprising any 100consecutive amino acids numbered from 1-100, 2-101, 3-102, 4-103, 5-104,6-105, 7-106, 8-107, 9-108, 10-109, 11-110, 12-111, 13-112, 14-113,15-114, 16-115, 17-116, 18-117, 19-118, 20-119, 21-120, 22-121, 23-122,24-123, 25-124, 26-125, 27-126, 28-127, 29-128, 30-129, 31-130, 32-131,33-132, 34-133, 35-134, 36-135, 37-136, 38-137, 39-138, 40-139, 41-140,42-141, 43-142, 44-143, 45-144, 46-145, 47-146, 48-147, 49-148, 50-149,51-150, 52-151, 53-152, 54-153, 55-154, 56-155, 57-156, 58-157, 59-158,60-159, 61-160, 62-161, 63-162, 64-163, 65-164, 66-165, 67-166, 68-167,69-168, 70-169, 71-170, 72-171, 73-172, 74-173, 75-174, 76-175, 77-176,78-177, 79-178, 80-179, 81-180, 82-181, 83-182, 84-183, 85-184, 86-185,87-186, 88-187, 89-188, 90-189, 91-190, 92-191, 93-192, 94-193, 95-194,96-195, 97-196, 98-197, 99-198, 100-199, 101-200, 102-201, 103-202,104-203, 105-204, 106-205, 107-206, 108-207, 109-208, 110-209, 111-210,112-211, 113-212, 114-213, 115-214, 116-215, 117-216, 118-217, 119-218,120-219, 121-220, 122-221, 123-222, 124-223, 125-224, 126-225, 127-226,128-227, 129-228, 130-229, 131-230, 132-231, 133-232, 134-233, 135-234,136-235, 137-236, 138-237, 139-238, 140-239, 141-240, 142-241, 143-242,144-243, 145-244, 146-245, 147-246, 148-247, 149-248, 150-249, 151-250,152-251, 153-252, 154-253, 155-254, 156-255, 157-256, 158-257, 159-258,160-259, 161-260, 162-261, 163-262, 164-263, 165-264, 166-265, 167-266,168-267, 169-268, 170-269, 171-270, 172-271, 173-272, 174-273, 175-274,176-275, 177-276, 178-277, 179-278, 180-279, 181-280, 182-281, 183-282,184-283, 185-284, 186-285, 187-286, 188-287, 189-288, 190-289, 191-290,192-291, 193-292, 194-293, 195-294, 196-295, 197-296, 198-297, 199-298,200-299, 201-300, 202-301, 203-302, 204-303, 205-304, 206-305, 207-306,208-307, 209-308, 210-309, 211-310, 212-311, 213-312, 214-313, 215-314,216-315, 217-316, 218-317, 219-318, 220-319, 221-320, 222-321, 223-322,224-323, 225-324, 226-325, 227-326, 228-327, 229-328, 230-329, 231-330,232-331, 233-332, 234-333, 235-334, 236-335, 237-336, 238-337, 239-338,240-339, 241-340, 242-341, 243-342, 244-343, 245-344, 246-345, 247-346,248-347, 249-348, 250-349, 251-350, 252-351, 253-352, 254-353, 255-354,256-355, 257-356, 258-357, 259-358, 260-359, 261-360, 262-361, 263-362,264-363, 265-364, 266-365, 267-366, 268-367, 269-368, 270-369, 271-370,272-371, 273-372, 274-373, 275-374, 276-375, 277-376, 278-377, 279-378,280-379, 281-380, 282-381, 283-382, 284-383, 285-384, 286-385, 287-386,288-387, 289-388, 290-389, 291-390, 292-391, 293-392, 294-393, 295-394,296-395, 297-396, 298-397, 299-398, 300-399, 301-400, 302-401, 303-402,304-403, 305-404, 306-405, 307-406, 308-407, 309-408, 310-409, 311-410,312-411, 313-412, 314-413, 315-414, 316-415, 317-416, 318-417, 319-418,320-419, 321-420, 322-421, 323-422, 324-423, 325-424, 326-425, 327-426,328-427, 329-428, 330-429, 331-430, 332-431, 333-432, 334-433, 335-434,336-435, 337-436, 338-437, 339-438, 340-439, 341-440, 342-441, 343-442,344-443, 345-444, 346-445, 347-446, 348-447, 349-448, 350-449, 351-450,352-451, 353-452, 354-453, 355-454, 356-455, 357-456, 358-457, 359-458,360-459, 361-460, 362-461, 363-462, 364-463, 365-464, 366-465, 367-466,368-467, 369-468, 370-469, 371-470, 372-471, 373-472, 374-473, 375-474,376-475, 377-476, 378-477, 379-478, 380-479, 381-480, 382-481, 383-482,384-483, 385-484, 386-485, 387-486, 388-487, 389-488, 390-489, 391-490,392-491, 393-492, 394-493, 395-494, 396-495, 397-496, 398-497, 399-498,400-499, 401-500, 402-501, 403-502, 404-503, 405-504, 406-505, 407-506,408-507, 409-508, 410-509, 411-510, 412-511, 413-512, 414-513, 415-514,416-515, 417-516, 418-517, 419-518, 420-519, 421-520, 422-521, 423-522,424-523, 425-524, 426-525, 427-526, 428-527, 429-528, 430-529, 431-530,432-531, 433-532, 434-533, 435-534, 436-535, 437-536, 438-537, 439-538,440-539, 441-540, 442-541, 443-542, 444-543, 445-544, 446-545, 447-546,448-547, 449-548, 450-549, 451-550, 452-551, 453-552, 454-553, 455-554,456-555, 457-556, 458-557, 459-558, 460-559, 461-560, 462-561, 463-562,464-563, 465-564, 466-565, 467-566, 468-567, 469-568, 470-569, 471-570,472-571, 473-572, 474-573, 475-574, 476-575, 477-576, 478-577, 479-578,480-579, 481-580, 482-581, 483-582, 484-583, 485-584, 486-585, 487-586,488-587, 489-588, 490-589, 491-590, 492-591, 493-592, 494-593, 495-594,496-595, 497-596, 498-597, 499-598, 500-599, 501-600, 502-601, 503-602,504-603, 505-604, 506-605, 507-606, 508-607, 509-608, 510-609, 511-610,512-611, 513-612, 514-613, 515-614, 516-615, 517-616, 518-617, 519-618,520-619, 521-620, 522-621, 523-622, 524-623, 525-624, 526-625, 527-626,528-627, 529-628, 530-629, 531-630, 532-631, 533-632, 534-633, 535-634,536-635, 537-636, 538-637, 539-638, 540-639, 541-640, 542-641, 543-642,544-643, 545-644, 546-645, 547-646, 548-647, 549-648, 550-649, 551-650,552-651, 553-652, 554-653, 555-654, 556-655, 557-656, 558-657, 559-658,560-659, 561-660, 562-661, 563-662, 564-663, 565-664, 566-665, 567-666,568-667, 569-668, 570-669, 571-670, 572-671, 573-672, 574-673, 575-674,576-675, 577-676, 578-677, 579-678, 580-679, 581-680, 582-681, 583-682,584-683, 585-684, 586-685, 587-686, 588-687, 589-688, 590-689, 591-690,592-691, 593-692, 594-693, 595-694, 596-695, 597-696, 598-697, 599-698,600-699, 601-700, 602-701, 603-702, 604-703, 605-704, 606-705, 607-706,608-707, 609-708, 610-709, 611-710, 612-711, 613-712, 614-713, 615-714,616-715, 617-716, 618-717, 619-718, 620-719, 621-720, 622-721, 623-722,624-723, 625-724, 626-725, 627-726, 628-727, 629-728, 630-729, 631-730,632-731, 633-732, 634-733, 635-734, 636-735, 637-736, 638-737, 639-738,640-739, 641-740, 642-741, 643-742, 644-743, 645-744, 646-745, 647-746,648-747, 649-748, 650-749, 651-750, 652-751, 653-752, 654-753, 655-754,656-755, 657-756, 658-757, 659-758, 660-759, 661-760, 662-761, 663-762,664-763, 665-764, 666-765, 667-766, 668-767, 669-768, 670-769, 671-770,672-771, 673-772, 674-773, 675-774, 676-775, 677-776, 678-777, 679-778,680-779, 681-780, 682-781, 683-782, 684-783, 685-784, 686-785, 687-786,and 688-787, wherein the numbering of amino acids comprising any onefragment is consistent with the polypeptide sequence of any one EVENnumbered SEQ ID of the Sequence listing.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast”, “equal to”, “equal to or less than”, “less than”, “at least______ but not greater than ______” or “from ______ to ______ ” aspecified size or specified N-terminal and/or C-terminal positions. Itis noted that all ranges used to describe any embodiment of the presentinvention are inclusive unless specifically set forth otherwise.

The present invention also provides for the exclusion of any individualfragment specified by N-terminal and C-terminal positions or of anyfragment specified by size in amino acid residues as described above. Inaddition, any number of fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above may beexcluded as individual species. Further, any number of fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above may make up a polypeptide fragment inany combination and may optionally include non-GENSET and GENSET-Relatedpolypeptide sequences as well.

The above polypeptide fragments of the present invention can beimmediately envisaged using the above description and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specification. Moreover, the above fragments need nothave a GENSET biological activity, although polypeptides having theseactivities are preferred embodiments of the invention, since they wouldbe useful, for example, in immunoassays, in epitope mapping, epitopetagging, as vaccines, and as molecular weight markers. The abovefragments may also be used to generate antibodies to a particularportion of the polypeptide. These antibodies can then be used inimmunoassays well known in the art to distinguish between human andnon-human cells and tissues or to determine whether cells or tissues ina biological sample are or are not of the same type which express thepolypeptides of the present invention.

It is noted that the above species of polypeptide fragments of thepresent invention may alternatively be described by the formula “a tob”; where “a” equals the N-terminal most amino acid position and “b”equals the C-terminal most amino acid position of the polynucleotide;and further where “a” equals an integer between 1 and the number ofamino acids of the polypeptide sequence of the present invention minus6, and where “b” equals an integer between 7 and the number of aminoacids of the polypeptide sequence of the present invention; and where“a” is an integer smaller then “b” by at least 6.

The present invention also provides for the exclusion of any species ofpolypeptide fragments of the present invention specified by 5′ and 3′positions or sub-genuses of polypeptides specified by size in aminoacids as described above. Any number of fragments specified by 5′ and 3′positions or by size in amino acids, as described above, may beexcluded.

Functional Definition

Domains

Preferred polynucleotide fragments of the invention comprise domains ofpolypeptides of the invention. Such domains may eventually compriselinear or structural motifs and signatures including, but not limitedto, leucine zippers, helix-turn-helix motifs, post-translationalmodification sites such as glycosylation sites, ubiquitination sites,alpha helices, and beta sheets, signal sequences encoding signalpeptides which direct the secretion of the encoded proteins, sequencesimplicated in transcription regulation such as homeoboxes, acidicstretches, enzymatic active sites, substrate binding sites, andenzymatic cleavage sites. Such domains may present a particularbiological activity such as DNA or RNA-binding, secretion of proteins,transcription regulation, enzymatic activity, substrate bindingactivity, etc.

In a preferred embodiment, domains comprise a number of amino acids thatis any integer between 6 and 1000. Domains may be synthesized using anymethods known to those skilled in the art, including those disclosedherein. Methods for determining the amino acids which make up a domainwith a particular biological activity include mutagenesis studies andassays to determine the biological activity to be tested.

Alternatively, the polypeptides of the invention may be scanned formotifs, domains and/or signatures in databases using any computer methodknown to those skilled in the art. Searchable databases include Prosite[Hofmann et al., (1999) Nucl. Acids Res. 27:215-219; Bucher and Bairoch(1994) Proceedings 2nd International Conference on Intelligent Systemsfor Molecular Biology. Altman et al, Eds., pp 53-61, AAAIPress, MenloPark], Pfam [Sonnhammer, et al., (1997) Proteins. 28(3):405-20; Henikoffet al., (2000) Electrophoresis 21(9):1700-6; Bateman et., (2000) NucleicAcids Res. 28(1):263-6], Blocks [Henikoff et al., (2000) Nucleic AcidsRes. 28(1):228-30], Print [Attwood et al., (1996) Nucleic Acids Res.24(1):182-8], Prodom [Sonnhammer and Kahn, (1994) Protein Sci.3(3):482-92; Corpet et al. (2000) Nucleic Acids Res. 28(1):267-9], Sbase[Pongor et al. (1993) Protein Eng. 6(4):391-5; Murvai et al., (2000)Nucleic Acids Res. 28(1):260-2], Smart [Schultz et al. (1998) Proc NatlAcad Sci USA 95, 5857-5864], Dali/FSSP [Holm and Sander (1996) NucleicAcids Res. 24(1):206-9, Holm and Sander (1997) Nucleic Acids Res.25(1):231-4 and Holm and Sander (1999) Nucleic Acids Res. 27(1):244-7],HSSP [Sander and Schneider (1991) Proteins. 9(1):56-68.], CATH [Orengoet al., (1997) Structure. 5(8):1093-108; Pearl et al., (2000) BiochemSoc Trans. 28(2):269-75], SCOP [Murzin et al., (1995) J Mol Biol.247(4):536-40; Lo Conte et al., (2000) Nucleic Acids Res. 28(1):257-9],COG [Tatusov et al. (1997), Science, 278, 631:637 and Tatusov et al.(2000), Nucleic Acids Res. 28(1):33-6], specific family databases andderivatives thereof [Nevill-Manning et al., (1998) Proc. Natl. Acad.Sci. USA. 95, 5865-5871; Yona, et al, (1999), Proteins. 37(3):360-78;Attwood et al., (2000) Nucleic Acids Res. 28(1):225-7], each of whichdisclosures are hereby incorporated by reference in their entireties.For a review on available databases, see issue 1 of volume 28 of NucleicAcid Research (2000), which disclosure is hereby incorporated byreference in its entirety.

Epitopes and Antibody Fusions:

A preferred embodiment of the present invention is directed toepitope-bearing polypeptides and epitope-bearing polypeptide fragments.These epitopes may be “antigenic epitopes” or both an “antigenicepitope” and an “immunogenic epitope”. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the polypeptide is the immunogen. On the other hand, a region ofpolypeptide to which an antibody binds is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of immunogenic epitopesof a protein generally is less than the number of antigenic epitopes[see, e.g., Geysen et al, (1984), Proc. Natl. Acad. Sci. U.S.A.81:3998-4002, which disclosure is hereby incorporated by reference inits entirety]. It is particularly noted that although a particularepitope may not be immunogenic, it is nonetheless useful sinceantibodies can be made to both immunogenic and antigenic epitopes. Whenthe antigen is a polypeptide, it is customary to classify epitopes asbeing linear (i.e., composed of a contiguous sequence of amino acidsrepeated along the polypeptide chain) or nonlinear (i.e., composed ofamino acids brought into proximity as a result of the folding of thepolypeptide chain). Nonlinear epitopes are also called “conformational”because they arise through the folding of the polypeptide chain into aparticular conformation, i.e., a distinctive 3-D shape.

An epitope can comprise as few as 3 amino acids in a spatialconformation, which is unique to the epitope. Generally an epitopeconsists of at least 6 such amino acids, and more often at least 8-10such amino acids. In preferred embodiment, antigenic epitopes comprise anumber of amino acids that is any integer between 3 and 50. Fragmentswhich function as epitopes may be produced by any conventional means[see, e.g., Houghten (1985), Proc. Natl. Acad. Sci. USA 82:5131-5135],also further described in U.S. Pat. No. 4,631,21, which disclosures arehereby incorporated by reference in their entireties. Methods fordetermining the amino acids which make up an epitope include x-raycrystallography, 2-dimensional nuclear magnetic resonance, and epitopemapping, e.g., the Pepscan method described by Geysen, et al. (1984);PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506,which disclosures are hereby incorporated by reference in theirentireties. Nonlinear epitopes are determined by methods such as proteinfootprinting (U.S. Pat. No. 5,691,448, which disclosure is herebyincorporated by reference in its entirety). Another example is thealgorithm of Jameson and Wolf, (1988), Comp. Appl. Biosci. 4:181-186(said reference incorporated by reference in its entirety). TheJameson-Wolf antigenic analysis, for example, may be performed using thecomputer program PROTEAN, using default parameters (Version 4.0 Windows,DNASTAR, Inc., 1228 South Park Street Madison, Wis.

All fragments of the polypeptides of the present invention, at least 6amino acids residues in length, are included in the present invention asbeing useful as antigenic linear epitopes. Amino acid residuescomprising other immunogenic epitopes may be determined by Jameson-Wolfanalysis, by other similar algorithms, or by in vivo testing for anantigenic response using the methods described herein or those known inthe art. Immunogenic epitopes predicted by algorithm analysis describeonly amino acid residues comprising linear epitopes predicted to havethe highest degree of immunogenicity. Polypeptides of the presentinvention that are not specifically described as immunogenic are notconsidered non-antigenic as they may be antigenic in vivo.Alternatively, the polypeptides are most likely antigenic in vitro usingmethods such as phage display.

Preferably, the epitope-containing polypeptide comprises a contiguousspan of at least 6, preferably at least 8 to 10, more preferably 12, 15,20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275,or 300 amino acids of a polypeptide of the present invention.

Nonlinear epitopes comprise more than one noncontiguous polypeptidesequence of at least one amino acid each. Such epitopes result fromnoncontiguous polypeptides brought into proximity by secondary,tertiary, or quaternary structural features. Therefore, the presentinvention encompasses isolated, purified, or recombinant polypeptidesand fragments thereof which comprise a nonlinear epitope. Preferredpolypeptides providing nonlinear epitopes are formed by a contiguoussurface of natively folded protein and are thus at least 10 amino acidsin length, further preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75,100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids of apolypeptide of the present invention, to the extent that a contiguousspan of these lengths is consistent with the lengths of said selectedsequence. Further preferred polypeptides comprise full-lengthpolypeptide sequences selected from the group consisting of thepolypeptide sequences of the Sequence Listing. Additionally, nonlinearepitopes may be formed by synthetic peptides that mimic an antigenicsite or contiguous surface normally presented on a protein in the nativeconformation. Therefore, preferred polypeptides providing nonlinearepitopes may be formed by synthetic proteins that comprise a combinationof at least 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75,100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids.

The epitope-bearing fragments of the present invention preferablycomprise 6 to 50 amino acids (i.e. any integer between 6 and 50,inclusive) of a polypeptide of the present invention. Also, included inthe present invention are antigenic fragments between the integers of 6and the full length GENSET sequence of the sequence listing. Allcombinations of sequences between the integers of 6 and the full-lengthsequence of a GENSET polypeptide are included. The epitope-bearingfragments may be specified by either the number of contiguous amino acidresidues (as a sub-genus) or by specific N-terminal and C-terminalpositions (as species) as described above for the polypeptide fragmentsof the present invention. Any number of epitope-bearing fragments of thepresent invention may also be excluded in the same manner.

Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies that specifically bind the epitope (see,Wilson et al., 1984; and Sutcliffe et al., (1983), Science. 219:660-666,which disclosures are hereby incorporated by reference in theirentireties). The antibodies are then used in various techniques such asdiagnostic and tissue/cell identification techniques, as describedherein, and in purification methods such as immunoaffinitychromatography.

Similarly, immunogenic epitopes can be used to induce antibodiesaccording to methods well known in the art (see, Sutcliffe et al.,supra; Wilson et al., supra; Chow et al., (1985), Proc. Natl. Acad. Sci.USA. 82:910-914; and Bittle et al., (1985), Virol 66:2347-2354, whichdisclosures are hereby incorporated by reference in their entireties). Apreferred immunogenic epitope includes the natural GENSET protein. Theimmunogenic epitopes may be presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting.).

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods (see, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al., supra). If in vivo immunization is used,animals may be immunized with free peptide; however, anti-peptideantibody titer may be boosted by coupling of the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH) ortetanus toxoid. For instance, peptides containing cysteine residues maybe coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibody,which can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins [see, e.g., EPA 0,394,827; and Traunecker et al.,(1988), Nature. 331:84-86, which disclosures are hereby incorporated byreference in their entireties]. Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion can also bemore efficient in binding and neutralizing other molecules thanmonomeric polypeptides or fragments thereof alone [see, e.g.,Fountoulakis et al., (1995) Biochem. 270:3958-3964, which disclosure ishereby incorporated by reference in its entirety]. Nucleic acidsencoding the above epitopes can also be recombined with a gene ofinterest as an epitope tag to aid in detection and purification of theexpressed polypeptide.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe present invention thereby effectively generating agonists andantagonists of the polypeptides. See, for example, U.S. Pat. Nos.5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, et al. (1997),Curr Opinion Biotechnol. 8:724-733; Harayama (1998), Trends Biotechnol.16(2): 76-82; Hansson et al., (1999), J. Mol. Biol. 287:265-276; andLorenzo and Blasco (1998) Biotechniques. 24(2):308-313. (Each of thesedocuments are hereby incorporated by reference). In one embodiment, oneor more components, motifs, sections, parts, domains, fragments, etc.,of coding polynucleotides of the invention, or the polypeptides encodedthereby may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

The present invention further encompasses any combination of thepolypeptide fragments listed in this section.

Antibodies

Definitions

The present invention further relates to antibodies and T-cell antigenreceptors (TCR), which specifically bind the polypeptides, and morespecifically, the epitopes of the polypeptides of the present invention.The antibodies of the present invention include IgG (including IgG1,IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM,and IgY. The term “antibody” (Ab) refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, where abinding domain is formed from the folding of variable domains of anantibody molecule to form three-dimensional binding spaces with aninternal surface shape and charge distribution complementary to thefeatures of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. As used herein, the term“antibody” is meant to include whole antibodies, including single-chainwhole antibodies, and antigen binding fragments thereof. In a preferredembodiment the antibodies are human antigen binding antibody fragmentsof the present invention include, but are not limited to, Fab,Fab′F(ab)2 and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. The antibodies may be from any animal originincluding birds and mammals. Preferably, the antibodies are human,murine, rabbit, goat, guinea pig, camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies, which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodiesthat are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,and trispecific or have greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., (1991),J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny et al., (1992), J. Immunol.148:1547-1553, which disclosures are hereby incorporated by reference intheir entireties.

Antibodies of the present invention may be described or specified interms of the epitope(s) or epitope-bearing portion(s) of a polypeptideof the present invention, which are recognized or specifically bound bythe antibody. The antibodies may specifically bind a complete proteinencoded by a nucleic acid of the present invention, or a fragmentthereof Therefore, the epitope(s) or epitope bearing polypeptideportion(s) may be specified as described herein, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues, orotherwise described herein (including the sequence listing). Antibodieswhich specifically bind any epitope or polypeptide of the presentinvention may also be excluded as individual species. Therefore, thepresent invention includes antibodies that specifically bind specifiedpolypeptides of the present invention, and allows for the exclusion ofthe same.

Thus, another embodiment of the present invention is a purified orisolated antibody capable of specifically binding to a polypeptide ofthe present invention. In one aspect of this embodiment, the antibody iscapable of binding to a linear epitope-containing polypeptide comprisingat least 6 consecutive amino acids, preferably at least 8 to 10consecutive amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, or 100 consecutive amino acids of a polypeptides of the presentinvention. In another aspect of this embodiment, the antibody is capableof binding to a nonlinear epitope-containing polypeptide comprising 10amino acids in length, further preferably 12, 15, 20, 25, 30, 35, 40,50, 60, 75, or 100 amino acids, further preferably, a contiguous surfaceof the native conformation of a polypeptide of the present application.Additionally, the antibody is capable of binding a nonlinear epitopepresented by a synthetic peptide designed to mimic a contiguous surfaceof the native conformation of a polypeptide of a sequence selected fromthe group consisting of GENSET polypeptides. Antibodies that bind linearepitopes may be used in combination with antibodies that bind nonlinearepitopes for instance, in assays that detect proper protein folding.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not specificallybind any other analog, ortholog, or homologue of the polypeptides of thepresent invention are included. Antibodies that do not bind polypeptideswith less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55%,and less than 50% identity (as calculated using methods known in the artand described herein, e.g., using FASTDB and the parameters set forthherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies, which only bind polypeptides encoded by polynucleotides,which hybridize to a polynucleotide of the present invention understringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms oftheir binding affinity. Preferred binding affinities include those witha dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M,5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M,5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and10⁻¹⁵M.

The invention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated GENSET protein or to a fragment orvariant thereof comprising an epitope of the mutated GENSET protein.

Preparation of Antibodies

The antibodies of the present invention may be prepared by any suitablemethod known in the art. Some of these methods are described in moredetail in the example entitled “Example 1: Preparation of AntibodyCompositions to the GENSET protein”. For example, a polypeptide of thepresent invention or an antigenic fragment thereof can be administeredto an animal in order to induce the production of sera containing“polyclonal antibodies”. As used herein, the term “monoclonal antibody”is not limited to antibodies produced through hybridoma technology butit rather refers to an antibody that is derived from a single clone,including eukaryotic, prokaryotic, or phage clone, and not the method bywhich it is produced. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technology.

Hybridoma techniques include those known in the art [see, e.g., Harlowand Lane, (1988) Antibodies A Laboratory Manual. Cold Spring HarborLaboratory. pp. 53-242; Hammerling (1981), Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y. 563-681; said references incorporatedby reference in their entireties]. Fab and F(ab′)2 fragments may beproduced, for example, from hybridoma-produced antibodies by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle, whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., (1995) J. Immunol Methods, 182:41-50; Ames et al., (1995), J.Immunol. Meth., 184:177-186.; Kettleborough et al., (1994), Eur. LImmunol., 24:952-958; Persic et al., (1997), Gene, 1879-81; Burton etal. (1994), Adv. Immunol., 57:191-280; PCT/GB91/01134; WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717,5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637,5,780,225, 5,658,727 and 5,733,743 (said references incorporated byreference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′F(ab)2 and F(ab′)2 fragments can also be employed using methods known inthe art such as those disclosed in WO 92/22324; Mullinax et al, (1992),BioTechniques. 12(6):864-869; and Sawai et al., (1995), AJRI 34:26-34;and Better et al., (1988) Science. 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., (1991), Meth. Enymol. 203:46-88; Shu, et al.,(1993), Proc. Natl. Acad. Sci. U.S.A. 90:7995-7999; and Skerra, et al.,(1988), Science 240:1038-1040, which disclosures are hereby incorporatedby reference in their entireties. For some uses, including in vivo useof antibodies in humans and in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. Methods forproducing chimeric antibodies are known in the art. See e.g., Morrison,(1985); Oi et al., (1986), BioTechniques 4:214; Gillies et al., (1989),J. Immunol Methods. 125:191-202; and U.S. Pat. No. 5,807,715, whichdisclosures are hereby incorporated by reference in their entireties.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing [EP 0 592 106; EP 0 519 596; Padlan(1991), Molec. Immunol. 28(4/5):489-498; Studnicka et al., (1994),Protein Engineering. 7(6):805-814; Roguska et al., (1994), Proc. Natl.Acad. Sci. U.S.A. 91:969-973], and chain shuffling (U.S. Pat. No.5,565,332), which disclosures are hereby incorporated by reference intheir entireties. Human antibodies can be made by a variety of methodsknown in the art including phage display methods described above. Seealso, U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; WO98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO91/10741 (said references incorporated by reference in theirentireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalent and non-covalentconjugations) to a polypeptide of the present invention. The antibodiesmay be specific for antigens other than polypeptides of the presentinvention. For example, antibodies of the present invention may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387, which disclosuresare hereby incorporated by reference in their entireties. Fusedantibodies may also be used to target the polypeptides of the presentinvention to particular cell types, either in vitro or in vivo, byfusing or conjugating the polypeptides of the present invention toantibodies specific for particular cell surface receptors. Antibodiesfused or conjugated to the polypeptides of the present invention mayalso be used in vitro immunoassays and purification methods usingmethods known in the art [see e.g., Harbor, et al. supra; WO 93/21232;EP 0 439 095; Naramura et al., (1994), Immunol. Lett. 39:91-99; U.S.Pat. No. 5,474,981; Gillies et al., (1992), Proc Natl Acad Sci USA89:1428-1432; Fell et al., (1991), J. Immunol. 146:2446-2452; saidreferences incorporated by reference in their entireties].

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivohalf-life of the polypeptides or for use in immunoassays using methodsknown in the art. The polypeptides may also be fused or conjugated tothe above antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi et al.,(1991), Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng, X. X., et al.(1995), J. Immunol. 154:5590-5600; and Vil, et al. (1992), Proc NatlAcad Sci US 89:11337-11341 (said references incorporated by reference intheir entireties).

Non-human animals or mammals, whether wild-type or transgenic, whichexpress a different species of GENSET than the one to which antibodybinding is desired, and animals which do not express GENSET (i.e. aGENSET knock out animal as described herein) are particularly useful forpreparing antibodies. GENSET knock out animals will recognize all ormost of the exposed regions of a GENSET protein as foreign antigens, andtherefore produce antibodies with a wider array of GENSET epitopes.Moreover, smaller polypeptides with only 10 to 30 amino acids may beuseful in obtaining specific binding to any one of the GENSET proteins.In addition, the humoral immune system of animals which produce aspecies of GENSET that resembles the antigenic sequence willpreferentially recognize the differences between the animal's nativeGENSET species and the antigen sequence, and produce antibodies to theseunique sites in the antigen sequence. Such a technique will beparticularly useful in obtaining antibodies that specifically bind toany one of the GENSET proteins.

A preferred embodiment of the invention is a method of specificallybinding an antibody or antibody fragment to a GENSET polypeptide. Thismethod comprises the step of contacting a GENSET polypeptide-specificantibody or fragment thereof with a GENSET polypeptide underantibody-binding conditions. Further included is a method ofspecifically binding an antibody or antibody fragment to an epitope,domain, or fragment of a GENSET polypeptide. This method may be used to,for example, detect, purify, or modify the activity of GENSETpolypeptides, as disussed herein.

Antibodies of the invention can be used to assay protein levels in atest sample or biological sample using methods known to those of skillin the art. Antibody-based methods useful for detecting protein includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as glucose oxidase, horseradishperoxidase, and alkaline phosphatase; radioisotopes, such as iodine(125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In),and technetium (99Tc); luminescent labels, such luminol, isolumino,theromatic acridinium ester, imidazole, acridinium salt, oxalate ester,luciferin, luciferase, and aequorin; and fluorescent labels, such asfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine.

Uses of Polynucleotides

Uses of Polynucleotides as Reagents

The polynucleotides of the present invention may be used as reagents inisolation procedures, diagnostic assays, and forensic procedures. Forexample, sequences from the GENSET polynucleotides of the invention maybe detectably labeled and used as probes to isolate other sequencescapable of hybridizing to them. In addition, sequences from the GENSETpolynucleotides of the invention may be used to design PCR primers to beused in isolation, diagnostic, or forensic procedures.

To Find Corresponding Genomic DNA Sequences

The GENSET cDNAs of the invention may also be used to clone sequenceslocated upstream of the cDNAs of the invention on the correspondinggenomic DNA. Such upstream sequences may be capable of regulating geneexpression, including promoter sequences, enhancer sequences, and otherupstream sequences which influence transcription or translation levels.Once identified and cloned, these upstream regulatory sequences may beused in expression vectors designed to direct the expression of aninserted gene in a desired spatial, temporal, developmental, orquantitative fashion.

Use of cDNAs or Fragments Thereof to Clone Upstream Sequences fromGenomic DNA

Sequences derived from polynucleotides of the inventions may be used toisolate the promoters of the corresponding genes using chromosomewalking techniques. In one chromosome walking technique, theGenomeWalker™ kit available from Clontechis used according to themanufacturer's instructions.

Identification of Promoters in Cloned Upstream Sequences

Once the upstream genomic sequences have been cloned and sequenced,prospective promoters and transcription start sites within the upstreamsequences may be identified by comparing the sequences upstream of thepolynucleotides of the inventions with databases containing knowntranscription start sites, transcription factor binding sites, orpromoter sequences.

In addition, promoters in the upstream sequences may be identified usingpromoter reporter vectors as follows. The expression of the reportergene will be detected when placed under the control of regulatory activepolynucleotide fragments or variants of the GENSET promoter regionlocated upstream of the first exon of the GENSET gene. Suitable promoterreporter vectors, into which the GENSET promoter sequences may be clonedinclude pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, orpEGFP-1 Promoter Reporter vectors available from Clontech, or pGL2-basicor pGL3-basic promoterless luciferase reporter gene vector from Promega.Briefly, each of these promoter reporter vectors include multiplecloning sites positioned upstream of a reporter gene encoding a readilyassayable protein such as secreted alkaline phosphatase, luciferase,beta-galactosidase, or green fluorescent protein. The sequences upstreamthe GENSET coding region are inserted into the cloning sites upstream ofthe reporter gene in both orientations and introduced into anappropriate host cell. The level of reporter protein is assayed andcompared to the level obtained from a vector which lacks an insert inthe cloning site. The presence of an elevated expression level in thevector containing the insert with respect to the control vectorindicates the presence of a promoter in the insert. If necessary, theupstream sequences can be cloned into vectors which contain an enhancerfor increasing transcription levels from weak promoter sequences. Asignificant level of expression above that observed with the vectorlacking an insert indicates that a promoter sequence is present in theinserted upstream sequence. Promoter sequence within the upstreamgenomic DNA may be further defined by site directed mutagenesis, linkerscanning analysis, or other techniques familiar to those skilled in theart.

The strength and the specificity of the promoter of each GENSET gene canbe assessed through the expression levels of a detectable polynucleotideoperably linked to the GENSET promoter in different types of cells andtissues. The detectable polynucleotide may be either a polynucleotidethat specifically hybridizes with a predefined oligonucleotide probe, ora polynucleotide encoding a detectable protein, including a GENSETpolypeptide or a fragment or a variant thereof. This type of assay iswell known to those skilled in the art and is described in U.S. Pat.Nos. 5,502,176; 5,266,488; the disclosures of which are incorporated byreference herein in their entirety. Some of the methods are discussed inmore detail elsewhere in the application.

The promoters and other regulatory sequences located upstream of thepolynucleotides of the inventions may be used to design expressionvectors capable of directing the expression of an inserted gene in adesired spatial, temporal, developmental, or quantitative manner. Apromoter capable of directing the desired spatial, temporal,developmental, and quantitative patterns may be selected using theresults of the expression analysis described herein. For example, if apromoter which confers a high level of expression in muscle is desired,the promoter sequence upstream of a polynucleotide of the inventionderived from an mRNA which is expressed at a high level in muscle may beused in the expression vector.

To Find Similar Sequences

Polynucleotides of the invention may be used to isolate and/or purifynucleic acids similar thereto using any methods well known to thoseskilled in the art including the techniques based on hybridization or onamplification described in this section. These methods may be used toobtain the genomic DNAs which encode the mRNAs from which the GENSETcDNAs are derived, mRNAs corresponding to GENSET cDNAs, or nucleic acidswhich are homologous to GENSET cDNAs or fragments thereof, such asvariants, species homologues or orthologs.

Hybridization-Based Methods

Techniques for identifying cDNA clones in a cDNA library which hybridizeto a given probe sequence are disclosed in Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual. (2ed., Cold Spring HarborLaboratory, Cold Spring Harbor, New York), and in Hames and Higgins(1985) Nucleic Acid Hybridization: A Practical Approach (Hames andHiggins Ed., IRL Press, Oxford), the disclosures of which areincorporated herein by reference in their entireties. The sametechniques may be used to isolate genomic DNAs.

A probe comprising at least 10 consecutive nucleotides from a GENSETcDNA or fragment thereof is labeled with a detectable label such as aradioisotope or a fluorescent molecule.

Techniques for labeling the probe are well known and includephosphorylation with polynucleotide kinase, nick translation, in vitrotranscription, and non radioactive techniques. The cDNAs or genomic DNAsin the library are transferred to a nitrocellulose or nylon filter anddenatured. After blocking of nonspecific sites, the filter is incubatedwith the labeled probe for an amount of time sufficient to allow bindingof the probe to cDNAs or genomic DNAs containing a sequence capable ofhybridizing thereto.

By varying the stringency of the hybridization conditions used toidentify cDNAs or genomic DNAs which hybridize to the detectable probe,cDNAs or genomic DNAs having different levels of identity to the probecan be identified and isolated as described below.

Stringent Conditions

“Stringent hybridization conditions” are defined as conditions in whichonly nucleic acids having a high level of identity to the probe are ableto hybridize to said probe. These conditions may be calculated asfollows:

For probes between 14 and 70 nucleotides in length the meltingtemperature (Tm) is calculated using the formula: Tm=81.5+16.6(log(Na+))+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:Tm=81.5+16.6(log(Na+))+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50%formamide. The formulas for SSC and Denhardt's solutions are listed inSambrook et al., 1986.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto nucleic acids containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 15-25° C. below the Tm. Preferably, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C.Preferably, for hybridizations in 50% formamide containing solutions,the hybridization is conducted at approximately 42° C.

Following hybridization, the filter is washed in 2×SSC, 0.1% SDS at roomtemperature for 15 minutes. The filter is then washed with 0.1×SSC, 0.5%SDS at room temperature for 30 minutes to 1 hour. Thereafter, thesolution is washed at the hybridization temperature in 0.1×SSC, 0.5%SDS. A final wash is conducted in 0.1×SSC at room temperature.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

Low and Moderate Conditions

Changes in the stringency of hybridization and signal detection areprimarily accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. The above procedure maythus be modified to identify nucleic acids having decreasing levels ofidentity to the probe sequence. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a sodium concentration ofapproximately 1M. Following hybridization, the filter may be washed with2×SSC, 0.5% SDS at the temperature of hybridization. These conditionsare considered to be “moderate” conditions above 50° C. and “low”conditions below 50° C. Alternatively, the hybridization may be carriedout in buffers, such as 6×SSC, containing formamide at a temperature of42° C. In this case, the concentration of formamide in the hybridizationbuffer may be reduced in 5% increments from 50% to 0% to identify cloneshaving decreasing levels of identity to the probe. Followinghybridization, the filter may be washed with 6×SSC, 0.5% SDS at 50° C.These conditions are considered to be “moderate” conditions above 25%formamide and “low” conditions below 25% formamide. cDNAs or genomicDNAs which have hybridized to the probe are identified byautoradiography or other conventional techniques.

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Consequently, the present invention encompasses methods of isolatingnucleic acids similar to the polynucleotides of the invention,comprising the steps of:

-   -   a) contacting a collection of cDNA or genomic DNA molecules with        a detectable probe comprising at least 12, 15, 18, 20, 23, 25,        28, 30, 35, 40 or 50 consecutive nucleotides of a polynucleotide        of the present invention under stringent, moderate or low        conditions which permit said probe to hybridize to at least a        cDNA or genomic DNA molecule in said collection;    -   b) identifying said cDNA or genomic DNA molecule which        hybridizes to said detectable probe; and    -   c) isolating said cDNA or genomic DNA molecule which hybridized        to said probe.        PCR-Based Methods

In addition to the above described methods, other protocols areavailable to obtain homologous cDNAs using GENSET cDNA of the presentinvention or fragment thereof as outlined in the following paragraphs.

cDNAs may be prepared by obtaining mRNA from the tissue, cell, ororganism of interest using mRNA preparation procedures utilizing polyAselection procedures or other techniques known to those skilled in theart. A first primer capable of hybridizing to the polyA tail of the mRNAis hybridized to the mRNA and a reverse transcription reaction isperformed to generate a first cDNA strand.

The term “capable of hybridizing to the polyA tail of said mRNA” refersto and embraces all primers containing stretches of thymidine residues,so-called oligo(dT) primers, that hybridize to the 3′ end of eukaryoticpoly(A)+mRNAs to prime the synthesis of a first cDNA strand. Techniquesfor generating said oligo (dT) primers and hybridizing them to mRNA tosubsequently prime the reverse transcription of said hybridized mRNA togenerate a first cDNA strand are well known to those skilled in the artand are described in Current Protocols in Molecular Biology, John Wileyand Sons, Inc. 1997 and Sambrook, et al., 1989. Preferably, said oligo(dT) primers are present in a large excess in order to allow thehybridization of all mRNA 3′ ends to at least one oligo (dT) molecule.The priming and reverse transcription steps are preferably performedbetween 37° C. and 55° C. depending on the type of reverse transcriptaseused. Preferred oligo(dT) primers for priming reverse transcription ofmRNAs are oligonucleotides containing a stretch of thymidine residues ofsufficient length to hybridize specifically to the polyA tail of mRNAs,preferably of 12 to 18 thymidine residues in length. More preferably,such oligo(T) primers comprise an additional sequence upstream of thepoly(dT) stretch in order to allow the addition of a given sequence tothe 5′ end of all first cDNA strands which may then be used tofacilitate subsequent manipulation of the cDNA. Preferably, this addedsequence is 8 to 60 residues in length. For instance, the addition of arestriction site in 5′ of cDNAs facilitates subcloning of the obtainedcDNA. Alternatively, such an added 5′ end may also be used to designprimers of PCR to specifically amplify cDNA clones of interest.

The first cDNA strand is then hybridized to a second primer. Anyappropriate polynucleotide fragment of the invention may be used. Thissecond primer contains at least 10 consecutive nucleotides of apolynucleotide of the invention. Preferably, the primer comprises atleast 10, 12, 15, 17, 18,20, 23, 25, or 28 consecutive nucleotides of apolynucleotide of the invention. In some embodiments, the primercomprises more than 30 nucleotides of a polynucleotide of the invention.If it is desired to obtain cDNAs containing the full protein codingsequence, including the authentic translation initiation site, thesecond primer used contains sequences located upstream of thetranslation initiation site. The second primer is extended to generate asecond cDNA strand complementary to the first cDNA strand.Alternatively, RT-PCR may be performed as described above using primersfrom both ends of the cDNA to be obtained.

The double stranded cDNAs made using the methods described above areisolated and cloned. The cDNAs may be cloned into vectors such asplasmids or viral vectors capable of replicating in an appropriate hostcell. For example, the host cell may be a bacterial, mammalian, avian,or insect cell.

Techniques for isolating mRNA, reverse transcribing a primer hybridizedto mRNA to generate a first cDNA strand, extending a primer to make asecond cDNA strand complementary to the first cDNA strand, isolating thedouble stranded cDNA and cloning the double stranded cDNA are well knownto those skilled in the art and are described in Current Protocols inMolecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook, et al.,1989.

Consequently, the present invention encompasses methods of making cDNAs.In a first embodiment, the method of making a cDNA comprises the stepsof:

-   -   a) contacting a collection of mRNA molecules from human cells        with a primer comprising at least 12, 15, 18, 20, 23, 25, 28,        30, 35, 40, or 50 consecutive nucleotides of a sequence selected        from the group consisting of the polynucleotide sequences        complementary to the polynucleotide sequences of the Sequence        Listing and those complementary to a human cDNA clone insert of        the deposited clone pool;    -   b) hybridizing said primer to an mRNA in said collection;    -   c) reverse transcribing said hybridized primer to make a first        cDNA strand from said mRNA;    -   d) making a second cDNA strand complementary to said first cDNA        strand; and    -   e) isolating the resulting cDNA comprising said first cDNA        strand and said second cDNA strand.

Another embodiment of the present invention is a purified cDNAobtainable by the method of the preceding paragraph. In one aspect ofthis embodiment, the cDNA encodes at least a portion of a humanpolypeptide.

In a second embodiment, the method of making a cDNA comprises the stepsof:

-   -   a) contacting a collection of mRNA molecules from human cells        with a first primer capable of hybridizing to the polyA tail of        said mRNA;    -   b) hybridizing said first primer to said polyA tail;    -   c) reverse transcribing said mRNA to make a first cDNA strand;    -   d) making a second cDNA strand complementary to said first cDNA        strand using at least one primer comprising at least 12, 15, 18,        20, 23, 25, 28, 30, 35, 40, or 50 consecutive nucleotides of a        sequence selected from the group consisting of polynucleotide        sequences of the Sequence Listing and those of human cDNA clone        inserts of the deposited clone pool; and    -   e) isolating the resulting cDNA comprising said first cDNA        strand and said second cDNA strand.

In another aspect of this method the second cDNA strand is made by:

-   -   a) contacting said first cDNA strand with a second primer        comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or        50 consecutive nucleotides of a sequence selected from the group        consisting of polynucleotide sequences of the Sequence Listing        and those of human cDNA clone inserts of the deposited clone        pool, and a third primer which sequence is fully included within        the sequence of said first primer;    -   b) performing a first polymerase chain reaction with said second        and third primers to generate a first PCR product;    -   c) contacting said first PCR product with a fourth primer,        comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or        50 consecutive nucleotides of said sequence selected from the        group consisting of polynucleotide sequences of the Sequence        Listing and those of human cDNA clone inserts of the deposited        clone pool, and a fifth primer, which sequence is fully included        within the sequence of said third primer, wherein said fourth        and fifth hybridize to sequences within said first PCR product;        and    -   d) performing a second polymerase chain reaction, thereby        generating a second PCR product.

Alternatively, the second cDNA strand may be made by contacting saidfirst cDNA strand with a second primer comprising at least 12, 15, 18,20, 23, 25, 28, 30, 35, 40, or 50 consecutive nucleotides of a sequenceselected from the group consisting of polynucleotide sequences of theSequence Listing and human cDNA clone inserts of the deposited clonepool, and a third primer which sequence is fully included within thesequence of said first primer and performing a polymerase chain reactionwith said second and third primers to generate said second cDNA strand.

Alternatively, the second cDNA strand may be made by:

-   -   a) contacting said first cDNA strand with a second primer        comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or        50 consecutive nucleotides of a sequence selected from the group        consisting of polynucleotide sequences of the Sequence Listing        and human cDNA clone inserts of the deposited clone pool;    -   b) hybridizing said second primer to said first strand cDNA; and    -   c) extending said hybridized second primer to generate said        second cDNA strand.

Another embodiment of the present invention is a purified cDNAobtainable by a method of making a cDNA of the invention. In one aspectof this embodiment, said cDNA encodes at least a portion of a humanpolypeptide.

Other Protocols

Alternatively, other procedures may be used for obtaining homologouscDNAs. In one approach, cDNAs are prepared from mRNA and cloned intodouble stranded phagemids as follows. The cDNA library in the doublestranded phagemids is then rendered single stranded by treatment with anendonuclease, such as the Gene II product of the phage F1 and anexonuclease [Chang et al., (1993) Gene 127:95-8, which disclosure ishereby incorporated by reference in its entirety]. A biotinylatedoligonucleotide comprising the sequence of a fragment of a known GENSETcDNA, genomic DNA or fragment thereof is hybridized to the singlestranded phagemids. Preferably, the fragment comprises at least 10, 12,15, 17, 18, 20, 23, 25, or 28 consecutive nucleotides of apolynucleotide of the present invention.

Hybrids between the biotinylated oligonucleotide and phagemids areisolated by incubating the hybrids with streptavidin coated paramagneticbeads and retrieving the beads with a magnet [Fry et al., (1992)Biotechniques, 13: 124-131, which disclosure is hereby incorporated byreference in its entirety]. Thereafter, the resulting phagemids arereleased from the beads and converted into double stranded DNA using aprimer specific for the GENSET cDNA or fragment used to design thebiotinylated oligonucleotide. Alternatively, protocols such as the GeneTrapper kit (Gibco BRL), which disclosure is which disclosure is herebyincorporated by reference in its entirety, may be used. The resultingdouble stranded DNA is transformed into bacteria. Homologous cDNAs tothe GENSET cDNA or fragment thereof sequence are identified by colonyPCR or colony hybridization.

As a Chromosome Marker

GENSET polynucleotides may be mapped to their chromosomal locationsusing any methods or techniques known to those skilled in the artincluding radiation hybrid (RH) mapping, PCR-based mapping andFluorescence in situ hybridization (FISH) mapping described below.

Radiation Hybrid Mapping

Radiation hybrid (RH) mapping is a somatic cell genetic approach thatcan be used for high resolution mapping of the human genome. [See, e.g.,Benham et al. (1989) Genomics 4:509-517 and Cox et al., (1990) Science250:245-250; and Schuler et al., (1996) Science 274:540-546], whichdisclosure is hereby incorporated by reference in its entirety.]

Mapping of cDNAs to Human Chromosomes Using PCR Techniques

GENSET cDNAs and genomic DNAs may be assigned to human chromosomes usingPCR based methodologies. In such approaches, oligonucleotide primerpairs are designed from the cDNA sequence to minimize the chance ofamplifying through an intron. Preferably, the oligonucleotide primersare 18-23 bp in length and are designed for PCR amplification. Thecreation of PCR primers from known sequences is well known to those withskill in the art. For a review of PCR technology see Erlich (1992),which disclosure is hereby incorporated by reference in its entirety.

PCR is used to screen a series of somatic cell hybrid cell linescontaining defined sets of human chromosomes for the presence of a givencDNA or genomic DNA. DNA is isolated from the somatic hybrids and usedas starting templates for PCR reactions using the primer pairs from theGENSET cDNAs or genomic DNAs. Only those somatic cell hybrids withchromosomes containing the human gene corresponding to the GENSET cDNAor genomic DNA will yield an amplified fragment. The GENSET cDNAs orgenomic DNAs are assigned to a chromosome by analysis of the segregationpattern of PCR products from the somatic hybrid DNA templates. Thesingle human chromosome present in all cell hybrids that give rise to anamplified fragment is the chromosome containing that GENSET cDNA orgenomic DNA. For a review of techniques and analysis of results fromsomatic cell gene mapping experiments, see Ledbetter et al, (1990)Genomics 6:475-481, which disclosure is hereby incorporated by referencein its entirety.

Mapping of cDNAs to Chromosomes Using Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) allows the GENSET cDNA orgenomic DNA to be mapped to a particular location on a given chromosome.The chromosomes to be used for fluorescence in situ hybridizationtechniques may be obtained from a variety of sources including cellcultures, tissues, or whole blood.

In a preferred embodiment, chromosomal localization of a GENSET cDNA orgenomic DNA is obtained by FISH as described by Cherif et al., (1990),“Simultaneous Localization of Cosmids and Chromosome R-Banding byFluorescence Microscopy: Application to Regional Mapping of HumanChromosome 11”, Proc. Natl. Acad. Sci. U.S.A., 87:6639-6643, whichdisclosure is hereby incorporated by reference in its entirety. Forchromosomal localization, fluorescent R-bands are obtained as previouslydescribed (Cherif, et al., 1990, supra).

Use of cDNAs to Construct or Expand Chromosome Maps

Once the GENSET cDNAs or genomic DNAs have been assigned to particularchromosomes using any technique known to those skilled in the art thoseskilled in the art, particularly those described herein, they maybeutilized to construct a high resolution map of the chromosomes on whichthey are located or to identify the chromosomes in a sample.

Chromosome mapping involves assigning a given unique sequence to aparticular chromosome as described above. Once the unique sequence hasbeen mapped to a given chromosome, it is ordered relative to otherunique sequences located on the same chromosome. One approach tochromosome mapping utilizes a series of yeast artificial chromosomes(YACs) bearing several thousand long inserts derived from thechromosomes of the organism from which the GENSET cDNAs or genomic DNAsare obtained. This approach is described in Nagaraja et al., (1997) “Xchromosome map at 75-kb STS resolution, revealing extremes ofrecombination and GC content”, Genome Res. March 1997;7(3):210-22, whichdisclosure is hereby incorporated by reference in its entirety.

Identification of Genes Associated with Hereditary Diseases or DrugResponse

This example illustrates an approach useful for the association ofGENSET cDNAs or genomic DNAs with particular phenotypic characteristics.In this example, a particular GENSET cDNA or genomic DNA is used as atest probe to associate that GENSET cDNA or genomic DNA with aparticular phenotypic characteristic.

GENSET cDNAs or genomic DNAs are mapped to a particular location on ahuman chromosome using techniques such as those described herein orother techniques known in the art. A search of Mendelian Inheritance inMan (V. McKusick, Mendelian Inheritance in Man; available on linethrough Johns Hopkins University Welch Medical Library) reveals theregion of the human chromosome which contains the GENSET cDNA or genomicDNA to be a very gene rich region containing several known genes andseveral diseases or phenotypes for which genes have not been identified.The gene corresponding to this GENSET cDNA or genomic DNA thus becomesan immediate candidate for each of these genetic diseases.

Cells from patients with these diseases or phenotypes are isolated andexpanded in culture. PCR primers from the GENSET cDNA or genomic DNA areused to screen genomic DNA, mRNA or cDNA obtained from the patients.GENSET cDNAs or genomic DNAs that are not amplified in the patients canbe positively associated with a particular disease by further analysis.Alternatively, the PCR analysis may yield fragments of different lengthswhen the samples are derived from an individual having the phenotypeassociated with the disease than when the sample is derived from ahealthy individual, indicating that the gene containing the cDNA may beresponsible for the genetic disease.

Uses of Polynucleotides in Recombinant Vectors

The present invention also relates to recombinant vectors including theisolated polynucleotides of the present invention, and to host cellsrecombinant for a polynucleotide of the invention, such as the abovevectors, as well as to methods of making such vectors and host cells andfor using them for production of GENSET polypeptides by recombinanttechniques.

Recombinant Vectors

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, which is either double-stranded orsingle-stranded, and which comprise at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism. The present inventionencompasses a family of recombinant vectors that comprise a regulatorypolynucleotide and/or a coding polynucleotide derived from either theGENSET genomic sequence or the cDNA sequence. Generally, a recombinantvector of the invention may comprise any of the polynucleotidesdescribed herein, including regulatory sequences, coding sequences andpolynucleotide constructs, as well as any GENSET primer or probe asdefined herein.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide derived from a GENSETgenomic sequence or a GENSET cDNA, for example any cDNA selected fromthe group consisting of polynucleotide sequences of the SequenceListing, those of human cDNA clone inserts of the deposited clone pool,variants and fragments thereof in a suitable cell host, thispolynucleotide being amplified at every time that the recombinant vectorreplicates.

A second preferred embodiment of the recombinant vectors according tothe invention comprises expression vectors comprising either aregulatory polynucleotide or a coding nucleic acid of the invention, orboth. Within certain embodiments, expression vectors are employed toexpress a GENSET polypeptide which can be then purified and, for examplebe used in ligand screening assays or as an immunogen in order to raisespecific antibodies directed against the GENSET protein. In otherembodiments, the expression vectors are used for constructing transgenicanimals and also for gene therapy. Expression requires that appropriatesignals are provided in the vectors, said signals including variousregulatory elements, such as enhancers/promoters from both viral andmammalian sources that drive expression of the genes of interest in hostcells. Dominant drug selection markers for establishing permanent,stable cell clones expressing the products are generally included in theexpression vectors of the invention, as they are elements that linkexpression of the drug selection markers to expression of thepolypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding a GENSET protein, preferably aGENSET protein with an amino acid sequence selected from the groupconsisting of polypeptide sequences of the Sequence Listing, thoseencoded by the human cDNA clone inserts of the deposited clone pool,variants and fragments thereof. The polynucleotides of the presentinvention may be used to express an encoded protein in a host organismto produce a beneficial effect. In such procedures, the encoded proteinmay be transiently expressed in the host organism or stably expressed inthe host organism. The encoded protein may have any of the activitiesdescribed herein. The encoded protein may be a protein which the hostorganism lacks or, alternatively, the encoded protein may augment theexisting levels of the protein in the host organism.

Some of the elements which can be found in the vectors of the presentinvention are described in further detail in the following sections.

General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may comprise a chromosomal, non-chromosomal,semi-synthetic and synthetic DNA. Such a recombinant vector can comprisea transcriptional unit comprising an assembly of:

-   -   (1) a genetic element or elements having a regulatory role in        gene expression, for example promoters or enhancers. Enhancers        are cis-acting elements of DNA, usually from about 10 to 300 bp        in length that act on the promoter to increase the        transcription.    -   (2) a structural or coding sequence which is transcribed into        mRNA and eventually translated into a polypeptide, said        structural or coding sequence being operably linked to the        regulatory elements described in (1); and    -   (3) appropriate transcription initiation and termination        sequences. Structural units intended for use in yeast or        eukaryotic expression systems preferably include a leader        sequence enabling extracellular secretion of translated protein        by a host cell. Alternatively, when a recombinant protein is        expressed without a leader or transport sequence, it may include        a N-terminal residue. This residue may or may not be        subsequently cleaved from the expressed recombinant protein to        provide a final product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsignals, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation signals may be used toprovide the required non-transcribed genetic elements.

The in vivo expression of a GENSET polypeptide of the present inventionmay be useful in order to correct a genetic defect related to theexpression of the native gene in a host organism, for the treatment orprevention of any disease or condition that can be treated or preventedby increasing the level of GENSET polypeptide expression, or to theproduction of a biologically inactive GENSET protein. Consequently, thepresent invention also comprises recombinant expression vectors mainlydesigned for the in vivo production of a GENSET polypeptide the presentinvention by the introduction of the appropriate genetic material in theorganism or the patient to be treated. This genetic material may beintroduced in vitro in a cell that has been previously extracted fromthe organism, the modified cell being subsequently reintroduced in thesaid organism, directly in vivo into the appropriate tissue.

Regulatory Elements

The suitable promoter regions used in the expression vectors accordingto the present invention are chosen taking into account the cell host inwhich the heterologous gene has to be expressed.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted. Promoter regions can beselected from any desired gene using, for example, CAT (chloramphenicoltransferase) vectors and more preferably pKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the Lacd, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trppromoters (EP 0036776), the polyhedrin promoter, or the p10 proteinpromoter from baculovirus (Kit Novagen) [Smith et al., (1983) Mol. Cell.Biol. 3:2156-2165; O'Reilly et al. (1992), “Baculovirus ExpressionVectors: A Laboratory Manual”, W.H. Freeman and Co., New York; whichdisclosures are hereby incorporated by reference in their entireties],the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.Selection of a convenient vector and promoter is well within the levelof ordinary skill in the art.

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. Also contemplated as an element of the expression cassetteis a terminator. These elements can serve to enhance message levels andto minimize read through from the cassette into other sequences.

Selectable Markers

Selectable markers confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. Coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

Preferred Vectors

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), andpGEM1 (Promega Biotec, Madison, Wis., USA). Large numbers of othersuitable vectors are known to those of skill in the art, andcommercially available, such as the following bacterial vectors: pQE70,pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK,pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Bacteriophage Vectors

The P1 bacteriophage vector may contain large inserts ranging from about80 to about 100 kb. The construction of P1 bacteriophage vectors such asp158 or p158/neo8 are notably described by Sternberg (1992) TrendsGenet. 8:1-16, and Sternberg (1994) Mamm. Genome. 5:397-404, whichdisclosure is hereby incorporated by reference in its entirety.Recombinant P1 clones comprising GENSET nucleotide sequences may bedesigned for inserting large polynucleotides of more than 40 kb [see,Linton et al., (1993) J. Clin. Invest. 92:3029-3037], which disclosureis hereby incorporated by reference in its entirety. To generate P1 DNAfor transgenic experiments, a preferred protocol is the protocoldescribed by McCormick et al., (1994) Genet. Anal. Tech. Appl. 11:158-164, which disclosure is hereby incorporated by reference in itsentirety. Briefly, E. coli (preferably strain NS3529) harboring the P1plasmid are grown overnight in a suitable broth medium containing 25μg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkalinelysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif.,USA), according to the manufacturer's instructions. The P1 DNA ispurified from the bacterial lysate on two Qiagen-tip 500 columns, usingthe washing and elution buffers contained in the kit. Aphenol/chloroform extraction is then performed before precipitating theDNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl,pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed byspectrophotometry.

When the goal is to express a P1 clone comprising GENSETpolypeptide-encoding nucleotide sequences in a transgenic animal,typically in transgenic mice, it is desirable to remove vector sequencesfrom the P1 DNA fragment, for example by cleaving the P1 DNA atrare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1insert is then purified from vector sequences on a pulsed-field agarosegel, using methods similar to those originally reported for theisolation of DNA from YACs [see, e. g., Schedl et al., (1993a), Nature,362: 258-261; Peterson et al., (1993), Proc. Natl. Acad. Sci. USA,90:7593-7597], which disclosures are hereby incorporated by reference intheir entireties. At this stage, the resulting purified insert DNA canbe concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit(Millipore, Bedford, Mass., USA—30,000 molecular weight limit) and thendialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μMEDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on amicrodyalisis membrane (type VS, 0.025 μM from Millipore). Theintactness of the purified P1 DNA insert is assessed by electrophoresison 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel andstaining with ethidium bromide.

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg, (1996), Medecine/Sciences, 12:47-55, orOhno et al., (1994) Science. 265:781-784, which disclosures are herebyincorporated by reference in their entireties. Another preferredrecombinant adenovirus according to this specific embodiment of thepresent invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) oran adenovirus of animal origin (French patent application No.FR-93.05954, which disclosure is hereby incorporated by reference in itsentirety). Further included in the present invention areademo-associated virus vectors.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. Particularly preferred retroviruses for thepreparation or construction of retroviral in vitro or in vitro genedelivery vehicles of the present invention include retroviruses selectedfrom the group consisting of Mink-Cell Focus Inducing Virus, MurineSarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.Particularly preferred Murine Leukemia Viruses include the 4070A and the1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross(ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine LeukemiaVirus (ATCC No VR-190; PCT Application No WO 94/24298). Particularlypreferred Rous Sarcoma Viruses include Bryan high titer (ATCC NosVR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviralvectors are those described in Roth et al., (1996) Nature Medicine,2(9):985-991, PCT Application No WO 93/25234, PCT Application No WO94/06920, Roux et al. (1989), Proc. Natl. Acad. Sci. U.S.A.86:9079-9083, Julan et al. (1992), J. Gen. Virol. 73:3251-3255, and Nedaet al. (1991), J. Biol. Chem. 266:14143-14146, which disclosures arehereby incorporated by reference in their entireties.

BAC Vectors

The bacterial artificial chromosome (BAC) cloning system [Shizuya et al.(1992), Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797], which disclosure ishereby incorporated by reference in its entirety, has been developed tostably maintain large fragments of genomic DNA (100-300 kb) in E. coli.A preferred BAC vector comprises a pBeloBAC11 vector that has beendescribed by Kim U-J. et al. (1996), Genomics 34:213-218, whichdisclosure is hereby incorporated by reference in its entirety. BAClibraries are prepared with this vector using size-selected genomic DNAthat has been partially digested using enzymes that permit ligation intoeither the Bam HI or HindIII sites in the vector. Flanking these cloningsites are T7 and SP6 RNA polymerase transcription initiation sites thatcan be used to generate end probes by either RNA transcription or PCRmethods. After the construction of a BAC library in E. coli, BAC DNA ispurified from the host cell as a supercoiled circle. Converting thesecircular molecules into a linear form precedes both size determinationand introduction of the BACs into recipient cells. The cloning site isflanked by two NotI sites, permitting cloned segments to be excised fromthe vector by NotI digestion. Alternatively, the DNA insert contained inthe pBeloBAC11 vector may be linearized by treatment of the BAC vectorwith the commercially available enzyme lambda terminate that leads tothe cleavage at the unique cosN site, but this cleavage method resultsin a full length BAC clone containing both the insert DNA and the BACsequences.

Baculovirus

Another specific suitable host vector system is the pVL1392/1393baculovirus transfer vector (Pharmingen) that is used to transfect theSF9 cell line (ATCC No. CRL 1711) which is derived from Spodopterafrugiperda. Other suitable vectors for the expression of the GENSETpolypeptide of the present invention in a baculovirus expression systeminclude those described by Chai et al. (1993), Biotechnol. Appl.Biochem. 18:259-273; Vlasak, et al. (1983), Eur. J. Biochem.135:123-126, and Lenhard et al., (1996) Gene. 169:187-190, whichdisclosures are hereby incorporated by reference in their entireties.

Delivery of the Recombinant Vectors

To effect expression of the polynucleotides and polynucleotideconstructs of the invention, the constructs must be delivered into acell. This delivery may be accomplished in vitro, as in laboratoryprocedures for transforming cell lines, or in vivo or ex vivo, as in thetreatment of certain diseases states.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle. The expression construct,preferably a recombinant viral vector as discussed herein, may transducepackaging cells through any means known in the art such aselectroporation, liposomes, and CaPO4 precipitation. The packaging cellgenerates infectious viral particles that include a polynucleotideencoding a polypeptide of the present invention. Such viral particlesthen may be employed to transduce eukaryotic cells in vitro, ex vivo orin vivo. The transduced eukaryotic cells will express a polypeptide ofthe present invention. Preferably, the viruses used in the presentinvention are rendered replication deficient by deletion of one or moreof all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1through L5 (U.S. Pat. No. 6,228,844, which disclosure is herebyincorporated by reference in its entirety). Viral delivery is discussedin more detail herein (see also, U.S. Pat. No. 5,968,821, whichdisclosure is hereby incorporated by reference in its entirety).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding oneof the subject CCR-proteins, rendering the retrovirus replicationdefective. The replication defective retrovirus is then packaged intovirions which can be used to infect a target cell through the use of ahelper virus by standard techniques.

Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Examples ofsuitable packaging virus lines for preparing both ecotropic andamphotropic retroviral systems include .psi.Crip, .psi.Cre, .psi.2 and.psi.Am. Retroviruses have been used to introduce a variety of genesinto many different cell types, including neural cells, epithelialcells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bonemarrow cells, in vitro and/or in vivo (see for example Eglitis, et al.(1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:6460-6464; Wilson, et al. (1988) Proc. Natl. Acad.Sci. USA 85:3014-3018; Armentano, et al. (1990) Proc. Natl. Acad. Sci.USA 87:6141-6145; Huber, et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury, et al. (1991) Science 254:1802-1805; vanBeusechem, et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay,et al. (1992) Human Gene Therapy 3:641-647; Dai, et al. (1992) Proc.Natl. Acad. Sci. USA 89:10892-10895; Hwu, et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux, et al. (1989) PNAS 86:9079-9083; Julan, et al. (1992) J.Gen Virol 73:3251-3255; and Goud, et al. (1983) Virology 163:251-254);or coupling cell surface ligands to the viral env proteins (Neda, et al.(1991) J Biol Chem 266:14143-14146). Coupling can be in the form of thechemical cross-linking with a protein or other variety (e.g. lactose toconvert the env protein to an asialoglycoprotein), as well as bygenerating fusion proteins (e.g. single-chain antibody/env fusionproteins). This technique, while useful to limit or otherwise direct theinfection to certain tissue types, and can also be used to convert anecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequencesthat control expression of the desired gene.

Another viral gene delivery system useful in the present inventionutilitizes adenovirus-derived vectors. The genome of an adenovirus canbe manipulated such that it encodes a gene product of interest, but isinactivate in terms of its ability to replicate in a normal lytic virallife cycle (see, for example, Berkner, et al. (1988) BioTechniques6:616; Rosenfeld, et al. (1991) Science 252:431-434; and Rosenfeld, etal. (1992) Cell 68:143-155). Suitable adenoviral vectors derived fromthe adenovirus strain Ad type 5 dl324 or other strains of adenovirus(e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Recombinant adenoviruses can be advantageous in certain circumstances inthat they are not capable of infecting nondividing cells and can be usedto infect a wide variety of cell types, including airway epithelium(Rosenfeld, et al. (1992) cited supra), endothelial cells (Lemarchand etal. (1992) Proc. Natl. Sci. USA 89:6482-6486), hepatocytes (Herz andGerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells(Quantin, et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralpolynucleotides (and foreign polynucleotides contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Haj-Ahmand and Graham (1986) J.Virol. 57:267). Most replication-defective adenoviral vectors currentlyin use and therefore favored by the present invention are deleted forall or parts of the viral E1 and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, e.g., Jones, et al. (1979) Cell16:683; Berkner, et al., supra; and Graham, et al. in Methods inMolecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol.7. pp.109-127). Expression of desired polynucleotides can be undercontrol of, for example, the E1A promoter, the major late promoter (MLP)and associated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Yet another viral vector system useful for delivery of polynucleotidesis the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka, etal., Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its nucleic acids intonon-dividing cells, and exhibits a high frequency of stable integration(see for example Flotte et al., (1992) Am. J. Respir. Cell Mol. Biol.7:349-356; Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al.(1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol.62:1963-1973). Vectors containing as little as 300 base pairs of AAV canbe packaged and can integrate. Space for exogenous DNA is limited toabout 4.5 kb. An AAV vector such as that described in Tratschin, et al.(1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA intocells. A variety of nucleic acids have been introduced into differentcell types using AAV vectors (see for example Hermonat, et al. (1984)Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin, et al. (1985) Mol.Cell. Biol. 4:2072-2081; Wondisford, et al. (1988) Mol. Endocrinol.2:32-39; Tratschin, et al. (1984) J. Virol. 51:611-619; and Flotte, etal. (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapyhave been derived from herpes virus, vaccinia virus, and several RNAviruses. In particular, herpes virus vectors may provide a uniquestrategy for persistence of inserted gene expression in cells of thecentral nervous system and ocular tissue (Pepose, et al. (1994) InvestOphthalmol Vis Sci 35:2662-2666).

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calciun phosphate precipitation[Graham et al., (1973) Virol. 52:456-457; Chen et al. (1987) Mol. Cell.Biol. 7:2745-2752]; DEAE-dextran [Gopal (1985) Mol. Cell. Biol.,5:1188-1190]; electroporation [Tur-Kaspa et al. (1986) Mol. Cell. Biol.6:716-718; Potter et al., (1984) Proc. Natl. Acad. Sci. U.S.A.81(22):7161-7165]; direct microinjection (Harland et al., (1985) J.Cell. Biol. 101: 1094-1095); DNA-loaded liposomes [Nicolau et al.,(1982) Biochim. Biophys. Acta. 721:185-190; Fraley et al., (1979) Proc.Natl. Acad. Sci. USA. 76:3348-3352]; and receptor-mediated transfection.[Wu and Wu (1987), J. Biol. Chem. 262:4429-4432; and Wu and Wu (1988),Biochemistry 27:887-892], which disclosures are hereby incorporated byreference in their entireties. Some of these techniques may besuccessfully adapted for in vivo or ex vivo use, as discussed herein.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No. WO 95/11307 (Institut Pasteur,INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.(1996), Nature Medicine. 2(8):888-892 and of Huygen et al., (1996)Nature Medicine. 2(8):893-898, which disclosures are hereby incorporatedby reference in their entireties.

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be accomplished with particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.,(1987) Nature 327:70-73, which disclosure is hereby incorporated byreference in its entirety. Liposomal preparations for use in the presentinvention include cationic (positively charged), anionic (negativelycharged) and neutral preparations. However, cationic liposomes areparticularly preferred because a tight charge complex can be formedbetween the cationic liposome and the polyanionic nucleic acid. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner, et al., Proc. Nat. Acad. Sci. USA (1987) 84:7413-7416,which is herein incorporated by reference); mRNA (Malone, et al., Proc.Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporatedby reference); and purified transcription factors (Debs et al., J. Biol.Chem. (1990) 265:10189-10192, which is herein incorporated byreference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner, et al., Proc.Nad Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Similarly, anionic and neutral liposomes are readily available, such asfrom AvantiPolar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol. Theliposomes can comprise multilamellar vesicles (MLVs), small unilamellarvesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs beingpreferred. The various liposome-nucleic acid complexes are preparedusing methods well known in the art (Straubinger, et al., Methods ofImmunology (1983), 101:512-527, which is herein incorporated byreference). For example, MLVs containing nucleic acid can be prepared bydepositing a thin film of phospholipid on the walls of a glass tube andsubsequently hydrating with a solution of the material to beencapsulated (U.S. Pat. No. 5,965,421, which disclosure is herebyincorporated by reference). Generally, the ratio of DNA to liposomeswill be from about 10:1 to about 1:10. Preferably, the ration will befrom about 5:1 to about 1:5. More preferably, the ratio will be about3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.Additionally, liposomes may be targeted to specific cell types byembedding a targeting moiety such as a member of a receptor-receptorligand pair into the lipid envelope of the vesicle. Useful targetingmoieties specifically bind cell surface ligands, for example, CD48 orthe SCF receptor on mast cells. Thus, anti-CD48 antibodies or SCF ligandare examples of useful mast cell-targeting moieties (U.S. Pat. No.6,177,433, U.S. Pat. No. 6,110,490, and P.C.T No. WO9704748, whichdisclosures are hereby incorporated by reference in their entireties).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome [Ghosh and Bacchawat, (1991), Targeting ofliposomes to hepatocytes, IN: Liver Diseases, Targeted diagnosis andtherapy using specific receptors and ligands. Eds., Marcel Dekeker, NewYork, pp. 87-104; Wong, et al. (1980), Gene. 10:87-94; Nicolau et al.,(1987), Meth. Enzymol., 149:157-76, which disclosures are herebyincorporated by reference in their entireties].

In a specific embodiment, the invention provides a composition for thein vivo production of the GENSET polypeptides described herein. Itcomprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said protein or polypeptide.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired GENSETpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

Secretion Vectors

Some of the GENSET cDNAs or genomic DNAs of the invention may also beused to construct secretion vectors capable of directing the secretionof the proteins encoded by genes inserted in the vectors. Such secretionvectors may facilitate the purification or enrichment of the proteinsencoded by genes inserted therein by reducing the number of backgroundproteins from which the desired protein must be purified or enriched.Exemplary secretion vectors are described below.

The secretion vectors of the present invention include a promotercapable of directing gene expression in the host cell, tissue, ororganism of interest. Such promoters include the Rous Sarcoma Viruspromoter, the SV40 promoter, the human cytomegalovirus promoter, andother promoters familiar to those skilled in the art.

A signal sequence from a polynucleotide of the invention and signalsequences of clone inserts of the deposited clone pool is operablylinked to the promoter such that the mRNA transcribed from the promoterwill direct the translation of the signal peptide. The host cell,tissue, or organism may be any cell, tissue, or organism whichrecognizes the signal peptide encoded by the signal sequence in theGENSET cDNA or genomic DNA. Suitable hosts include mammalian cells,tissues or organisms, avian cells, tissues, or organisms, insect cells,tissues or organisms, or yeast.

In addition, the secretion vector contains cloning sites for insertinggenes encoding the proteins which are to be secreted. The cloning sitesfacilitate the cloning of the insert gene in frame with the signalsequence such that a fusion protein in which the signal peptide is fusedto the protein encoded by the inserted gene is expressed from the mRNAtranscribed from the promoter. The signal peptide directs theextracellular secretion of the fusion protein.

The secretion vector may be DNA or RNA and may integrate into thechromosome of the host, be stably maintained as an extrachromosomalreplicon in the host, be an artificial chromosome, or be transientlypresent in the host. Preferably, the secretion vector is maintained inmultiple copies in each host cell. As used herein, multiple copies meansat least 2, 5, 10, 20, 25, 50 or more than 50 copies per cell. In someembodiments, the multiple copies are maintained extrachromosomally. Inother embodiments, the multiple copies result from amplification of achromosomal sequence.

Many nucleic acid backbones suitable for use as secretion vectors areknown to those skilled in the art, including retroviral vectors, SV40vectors, Bovine Papilloma Virus vectors, yeast integrating plasmids,yeast episomal plasmids, yeast artificial chromosomes, human artificialchromosomes, P element vectors, baculovirus vectors, or bacterialplasmids capable of being transiently introduced into the host.

The secretion vector may also contain a polyA signal such that the polyAsignal is located downstream of the gene inserted into the secretionvector.

After the gene encoding the protein for which secretion is desired isinserted into the secretion vector, the secretion vector is introducedinto the host cell, tissue, or organism using calcium phosphateprecipitation, DEAE-Dextran, electroporation, liposome-mediatedtransfection, viral particles or as naked DNA. The protein encoded bythe inserted gene is then purified or enriched from the supernatantusing conventional techniques such as ammonium sulfate precipitation,immunoprecipitation, immunochromatography, size exclusionchromatography, ion exchange chromatography, and hplc. Alternatively,the secreted protein may be in a sufficiently enriched or pure state inthe supernatant or growth media of the host to permit it to be used forits intended purpose without further enrichment.

The signal sequences may also be inserted into vectors designed for genetherapy. In such vectors, the signal sequence is operably linked to apromoter such that mRNA transcribed from the promoter encodes the signalpeptide. A cloning site is located downstream of the signal sequencesuch that a gene encoding a protein whose secretion is desired mayreadily be inserted into the vector and fused to the signal sequence.The vector is introduced into an appropriate host cell. The proteinexpressed from the promoter is secreted extracellularly, therebyproducing a therapeutic effect.

Cell Hosts

Another object of the invention comprises a host cell that has beentransformed or transfected with one of the polynucleotides describedherein, and in particular a polynucleotide either comprising a GENSETpolypeptide-encoding polynucleotide regulatory sequence or thepolynucleotide coding for a GENSET polypeptide. Also included are hostcells that are transformed (prokaryotic cells), transfected (eukaryoticcells), or transduced with a recombinant vector such as one of thosedescribed above. However, the cell hosts of the present invention cancomprise any of the polynucleotides of the present invention. Preferredhost cells used as recipients for the expression vectors of theinvention are the following:

-   -   a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-α        strain), Bacillus subtilis, Salmonella typhimurium, and strains        from species like Pseudomonas, Streptomyces and Staphylococcus.    -   b) Eukaryotic host cells: HeLa cells (ATCC No.CCL2; No.CCL2.1;        No.CCL2.2), Cv 1 cells (ATCC No.CCL70), COS cells (ATCC No.        CRL1650; No. CRL1651), Sf-9 cells (ATCC No.CRL1711), C127 cells        (ATCC No. CRL-1804), 3T3 (ATCC No. CRL-6361), CHO (ATCC No.        CCL-61), human kidney 293. (ATCC No. 45504; No. CRL-1573) and        BHK (ECACC No. 84100501; No. 84111301).

The present invention also encompasses primary, secondary, andimmortalized homologously recombinant host cells of vertebrate origin,preferably mammalian origin and particularly human origin, that havebeen engineered to: a) insert exogenous (heterologous) polynucleotidesinto the endogenous chromosomal DNA of a targeted gene, b) deleteendogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNAwith exogenous polynucleotides. Insertions, deletions, and/orreplacements of polynucleotide sequences may be to the coding sequencesof the targeted gene and/or to regulatory regions, such as promoter andenhancer sequences, operably associated with the targeted gene.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination, see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller, et al.,(1989); and Zijlstra, et al. (1989) (the disclosures of each of whichare incorporated by reference in their entireties).

The present invention further relates to a method of making ahomologously recombinant host cell in vitro or in vivo, wherein theexpression of a targeted gene not normally expressed in the cell isaltered. Preferably the alteration causes expression of the targetedgene under normal growth conditions or under conditions suitable forproducing the polypeptide encoded by the targeted gene. The methodcomprises the steps of: (a) transfecting the cell in vitro or in vivowith a polynucleotide construct, said polynucleotide constructcomprising; (i) a targeting sequence; (ii) a regulatory sequence and/ora coding sequence; and (iii) an unpaired splice donor site, ifnecessary, thereby producing a transfected cell; and (b) maintaining thetransfected cell in vitro or in vivo under conditions appropriate forhomologous recombination.

The present invention further relates to a method of altering theexpression of a targeted gene in a cell in vitro or in vivo wherein thegene is not normally expressed in the cell, comprising the steps of: (a)transfecting the cell in vitro or in vivo with a polynucleotideconstruct, said polynucleotide construct comprising: (i) a targetingsequence; (ii) a regulatory sequence and/or a coding sequence; and (iii)an unpaired splice donor site, if necessary, thereby producing atransfected cell; and (b) maintaining the transfected cell in vitro orin vivo under conditions appropriate for homologous recombination,thereby producing a homologously recombinant cell; and (c) maintainingthe homologously recombinant cell in vitro or in vivo under conditionsappropriate for expression of the gene.

The present invention further relates to a method of making apolypeptide of the present invention by altering the expression of atargeted endogenous gene in a cell in vitro or in vivo wherein the geneis not normally expressed in the cell, comprising the steps of: a)transfecting the cell in vitro with a polynucleotide construct, saidpolynucleotide construct comprising: (i) a targeting sequence; (ii) aregulatory sequence and/or a coding sequence; and (iii) an unpairedsplice donor site, if necessary, thereby producing a transfected cell;(b) maintaining the transfected cell in vitro or in vivo underconditions appropriate for homologous recombination, thereby producing ahomologously recombinant cell; and c) maintaining the homologouslyrecombinant cell in vitro or in vivo under conditions appropriate forexpression of the gene thereby making the polypeptide.

The present invention further relates to a polynucleotide constructwhich alters the expression of a targeted gene in a cell type in whichthe gene is not normally expressed. This occurs when the polynucleotideconstruct is inserted into the chromosomal DNA of the target cell,wherein said polynucleotide construct comprises: a) a targetingsequence; b) a regulatory sequence and/or coding sequence; and c) anunpaired splice-donor site, if necessary. Further included are apolynucleotide construct, as described above, wherein saidpolynucleotide construct further comprises a polynucleotide whichencodes a polypeptide and is in-frame with the targeted endogenous geneafter homologous recombination with chromosomal DNA.

The compositions may be produced, and methods performed, by techniquesknown in the art, such as those described in U.S. Pat. Nos. 6,054,288;6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734;International Publication NOs: WO96/29411, WO 94/12650; and scientificarticles described by Koller, et al., (1994). (The disclosures of eachof which are incorporated by reference in their entireties.)

GENSET gene expression in mammalian cells, preferably human cells, maybe rendered defective, or alternatively may be altered by replacingendogenous GENSET polypeptide-encoding genes in the genome of an animalcell by a GENSET polypeptide-encoding polynucleotide according to theinvention. These genetic alterations may be generated by homologousrecombination using previously described specific polynucleotideconstructs.

Mammal zygotes, such as murine zygotes may be used as cell hosts. Forexample, murine zygotes may undergo microinjection with a purified DNAmolecule of interest.

Any one of the polynucleotides of the invention, including thePolynucleotide constructs described herein, may be introduced in anembryonic stem (ES) cell line, preferably a mouse ES cell line. ES celllines are derived from pluripotent, uncommitted cells of the inner cellmass of pre-implantation blastocysts. Preferred ES cell lines are thefollowing: ES-E14TG2a (ATCC No.CRL-1821), ES-D3 (ATCC No.CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). EScells are maintained in an uncommitted state by culture in the presenceof growth-inhibited feeder cells which provide the appropriate signalsto preserve this embryonic phenotype and serve as a matrix for ES celladherence. Preferred feeder cells are primary embryonic fibroblasts thatare established from tissue of day 13- day 14 embryos of virtually anymouse strain, that are maintained in culture, such as described byAbbondanzo et al., (1993), Meth. Enzymol., Academic Press, New York, pp803-823 and are growth-inhibited by irradiation, such as described byRobertson, (1987), Embryo-derived stem cell lines; In: E. J. RobertsonEd. Teratocarcinomas and embrionic stem cells: a practical approach. IRLPress, Oxford, pp. 71, or by the presence of an inhibitory concentrationof LIF, such as described by Pease and William, (1990), Exp. Cell. Res.190: 209-211, which disclosures are hereby incorporated by reference intheir entireties.

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Transgenic Animals

The terms “transgenic animals” or “host animals” are used herein todesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. The cells affected may be somactic, germ cells, or both.Preferred animals are non-human mammals and include those belonging to agenus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus(e.g. rabbits) which have their genome artificially and geneticallyaltered by the insertion of a nucleic acid according to the invention.In one embodiment, the invention encompasses non-human host mammals andanimals comprising a recombinant vector of the invention or a GENSETgene disrupted by homologous recombination with a knock out vector.

The transgenic animals of the invention all include within a pluralityof their cells a cloned recombinant or synthetic DNA sequence, morespecifically one of the purified or isolated nucleic acids comprising aGENSET polypeptide coding sequence, a GENSET polynucleotide regulatorysequence, a polynucleotide construct, or a DNA sequence encoding anantisense polynucleotide such as described in the present specification.

Generally, a transgenic animal according the present invention comprisesany of the polynucleotides, the recombinant vectors and the cell hostsdescribed in the present invention. In a first preferred embodiment,these transgenic animals may be good experimental models in order tostudy the diverse pathologies related to the dysregulation of theexpression of a given GENSET gene, in particular the transgenic animalscontaining within their genome one or several copies of an insertedpolynucleotide encoding a native GENSET polypeptide, or alternatively amutant GENSET polypeptide.

In a second preferred embodiment, these transgenic animals may express adesired polypeptide of interest under the control of the regulatorypolynucleotides of the GENSET gene, leading to high yields in thesynthesis of this protein of interest, and eventually to tissue specificexpression of the protein of interest.

The design of the transgenic animals of the invention may be madeaccording to the conventional techniques well known from the one skilledin the art. For more details regarding the production of transgenicanimals, and specifically transgenic mice, it may be referred to U.S.Pat. Nos. 4,873,191, issued Oct. 10, 1989; 5,464,764 issued Nov. 7,1995; and 5,789,215, issued Aug. 4, 1998; these documents being hereinincorporated by reference to disclose methods producing transgenic mice.

Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thathas incorporated exogenous genetic material. The procedure involvesobtaining the genetic material which encodes either a GENSET polypeptidecoding sequence, a GENSET polynucleotide regulatory sequence, or a DNAsequence encoding a GENSET polynucleotide antisense sequence, or aportion thereof, such as described in the present specification. Arecombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. [See, e.g., Thomas, et al. (1987) Cell.51:503-512, which disclosure is hereby incorporated by reference in itsentirety.] An illustrative positive-negative selection procedure thatmay be used according to the invention is described by Mansour et al.,(1988) Nature. 336:348-352, which disclosure is hereby incorporated byreference in its entirety.

The positive cells are then isolated, cloned and injected into 3.5 daysold blastocysts from mice, such as described by Bradley (1987)[Production and analysis of chimaeric mice In: E. J. Robertson (Ed.),Teratocarcinomas and embryonic stem cells: A practical approach. IRLPress, Oxford, pp.113)], which disclosure is hereby incorporated byreference in its entirety. The blastocysts are then inserted into afemale host animal and allowed to grow to term. Alternatively, thepositive ES cells are brought into contact with embryos at the 2.5 daysold 8-16 cell stage (morulae) such as described by Wood, et al. (1993),Proc. Natl. Acad. Sci. USA, 90: 4582-4585, or by Nagy et al., (1993),Proc. Natl. Acad. Sci. USA 90: 8424-8428, which disclosures are herebyincorporated by reference in their entireties, the ES cells beinginternalized to colonize extensively the blastocyst including the cellswhich will give rise to the germ line.

The offspring of the female host are tested to determine which animalsare transgenic e.g. include the inserted exogenous DNA sequence andwhich ones are wild type.

Thus, the present invention also concerns a transgenic animal containinga nucleic acid, a recombinant expression vector or a recombinant hostcell according to the invention.

In another embodiment, transgenic animals are produced by microinjectingpolynucleotides ares microinjected into a fertilized oocyte. Methods forculturing fertilized oocytes to the pre-implantation stage aredescribed, e.g., by Gordon, et al. ((1984) Methods in Enzymology, 101,414); Hogan, et al. [(1986) in Manipulating the mouse embryo, ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y (for the mouse embryo)]; Hammer, et al. [(1985) Nature, 315,680 (for rabbit and porcine embryos)]; Gandolfi, et al. [(1987) J.Reprod. Fert. 81, 23-28]; Rexroad, et al. [(1988) J. Anim. Sci.66,947-953) (for ovine embryos)]; and Eyestone, et al. [(1989) J.Reprod. Fert. 85, 715-720]; Camous et al. [(1984) J. Reprod. Fert. 72,779-785]; and Heyman, et al. [(1987) Theriogenology 27, 5968 (for bovineembryos)]; the disclosures of each of which are incorporated herein intheir entireties. Pre-implantation embryos are then transferred to anappropriate female by standard methods to permit the birth of atransgenic or chimeric animal, depending upon the stage of developmentwhen the transgene is introduced.

Any of a number of methods known in the art can be used to detect thepresence of a transgene in a pre-implantation embryo.

In a particularly preferred embodiment of the present invention,transgenic mammals are generated that secrete recombinant GENSETpolypeptides in their milk. As the mammary gland is a highly efficientprotein-producing organ, such methods can be used to produce proteinconcentrations in the gram per liter range, and often significantlymore. Preferably, expression in the mammary gland is accomplished byoperably linking the polynucleotide encoding the GENSET polypeptide to amammary gland specific promoter and, optionally, other regulatoryelements. Suitable promoters and other elements include, but are notlimited to, those derived from mammalian short and long WAP, alpha,beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes,as well as the the mouse mammary tumor virus (MMTV) promoter. Suchpromoters and other elements may be derived from any mammal, including,but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guineapigs. Promoter and other regulatory sequences, vectors, and otherrelevant teachings are provided, e.g., by Clark (1998) J Mammary GlandBiol Neoplasia 3:337-50; Jost, et al. (1999) Nat. Biotechnol 17:160-4;U.S. Pat. Nos. 5,994,616; 6,140,552; 6,013,857; Sohn, et al. (1999) DNACell Biol. 18:845-52; Kim, et al. (1999) J. Biochem. (Japan) 126:320-5;Soulier, et al. (1999) Euro. J. Biochem. 260:533-9; Zhang, et al. (1997)Chin. J. Biotech. 13:271-6; Rijnkels, et al. (1998) Transgen. Res.7:5-14; Korhonen, et al. (1997) Euro. J. Biochem. 245:482-9;Uusi-Oukari, et al. (1997) Transgen. Res. 6:75-84; Hitchin, et al.(1996) Prot. Expr. Purif. 7:247-52; Platenburg, et al. (1994) Transgen.Res. 3:99-108; Heng-Cherl, et al. (1993) Animal Biotech. 4:89-107; andChrista, et al. (2000) Euro. J. Biochem. 267:1665-71; the entiredisclosure of each of which is herein incorporated by reference.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens, etal. (2000) Biochim. Biophys. Acta 1523:161-171), in association with aligand that can lead to receptor-mediated endocytosis of the construct(see, e.g., Sobolev, et al. (1998) 273:7928-33), or in a viral vectorsuch as a retroviral vector, e.g. the Gibbon ape leukemia virus (see,e.g., Archer, et al. (1994) PNAS 91:6840-6844). In any of theseembodiments, the polynucleotide may be operably linked to a mammarygland specific promoter, as described above, or, alternatively, anystrongly expressing promoter such as CMV or MoMLV LTR.

The suitability of any vector, promoter, regulatory element, etc. foruse in the present invention can be assessed beforehand by transfectingcells such as mammary epithelial cells, e.g. MacT cells (bovine mammaryepithelial cells) or GME cells (goat mammary epithelial cells), in vitroand assessing the efficiency of transfection and expression of thetransgene in the cells.

In a preferred embodiment, a retroviral vector such as as Gibbon apeleukemia viral vector is used, as described in Archer, et al. ((1994)PNAS 91:6840-6844). As retroviral infection typically requires celldivision, cell division in the mammary glands can be stimulated inconjunction with the administration of the vector, e.g. using a factorsuch as estrodiol benzoate, progesterone, reserpine, or dexamethasone.Further, retroviral and other methods of infection can be facilitatedusing accessory compounds such as polybrene. Alternatively, anadenoviral or adeno-associated viral vector may be used to infectnon-dividing cells as discussed herein.

In any of the herein-described methods for obtaining GENSET polypeptidesfrom milk, the quantity of milk obtained, and thus the quantity ofGENSET polypeptides produced, can be enhanced using any standard methodof lacation induction, e.g. using hexestrol, estrogen, and/orprogesterone.

The polynucleotides used in such embodiments can either encode afull-length GENSET protein or a GENSET fragment. Typically, the encodedpolypeptide will include a signal sequence to ensure the secretion ofthe protein into the milk.

Recombinant Cell Lines Derived from the Transgenic Animals of theInvention:

A further object of the invention comprises recombinant host cellsobtained from a transgenic animal described herein. In one embodimentthe invention encompasses cells derived from non-human host mammals andanimals comprising a recombinant vector of the invention or a GENSETgene disrupted by homologous recombination with a knock out vector.Recombinant cell lines may be established in vitro from cells obtainedfrom any tissue of a transgenic animal according to the invention, forexample by transfection of primary cell cultures with vectors expressingonc-genes such as SV40 large T antigen, as described by Chou, (1989),Mol. Endocrinol. 3:1511-1514, and Shay et al., (1991), Biochem. Biophys.Acta, 1072: 1-7, which disclosures are hereby incorporated by referencein their entireties.

Uses of Polypeptides of the Invention

Protein of SEQ ID NO:24 (Internal Designation Clone 47-14-1-C3-CL0_(—)5)

The cDNA of clone 47-14-1-C3-CL0_(—)5 (SEQ ID NO:23) encodes the proteinof SEQ ID NO:24, comprising the amino acid sequence:

MVPFIYLQAHFTLCSGWSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSPEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWRCLRCLRQQHDDFADDISLLK. Accordingly, it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:24 described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in Clone47-14-1-C3-CL0_(—)5. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:23described throughout the present application also pertain to the nucleicacids included in Clone 47-14-1-C3-CL0_(—)5. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:23, SEQID NO:24, and Clone 47-14-1-C3-CL0_(—)5. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding the fragments.

Further preferred are compositions comprising the amino acid sequence:

SPEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAY;DLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCW Q andAITESDKFFINGSNWEGILGLAYAEIARPDDSPEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACH. Also preferred are polypeptide fragments having abiological activity as described herein and the polynucleotides encodingthe fragments.

The protein of SEQ ID NO:24 encodes amyloid processing inhibitor protein(APIP). APIP is expressed in mammalian tissues, particularly in neuronalcells, and is an incomplete aspartyl protease which is able to bindsubstrate but lacks catalytic activity. Examples of compounds whichinteract with APIP include, but are not limited to, amyloid betaprecursor protein, amyloid precursor like protein-1, amyloid precursorlike protein-2, Protease nexin-2, Anti-trypsin protein, Kunitz proteaseinhibitors and amyloid like proteins.

Amyloid beta precursor protein (APP) can be processed by several typesof proteases to yield fragments that are soluble or insoluble (Nunan andSmall, FEBS Lett (2000) 483(1):6-10, which disclosures are herebyincorporated by reference in their entirety). Sequential cleavage of APPby beta secretase and gamma secretase yields a secreted and insolublefibrillar amyloid protein, known as beta amyloid, which is the majorcomponent of extracellular amyloid plaques. Deposition of beta amyloidproteins form intraneuronal neurofibrillary tangles, amyloid plaques andvascular amyloid deposits characteristic of both Alzheimer's Disease andaged Down's Syndrome. Defects in processing APP can also lead tocerebral hemorraghage. Polypeptides of SEQ ID NO:24 and fragmentsthereof, bind to APP and other amyloid like proteins, and reduce therate of processing of these proteins.

In a number of embodiments, APIP is used to bind to and/or inhibit anyof a number of substrates in a biological sample. For example, onepreferred embodiment is directed to a method of contacting compositionscomprising APIP with APP. Further preferred is a method of contactingcompositions comprising APIP with amyloid precursor like protein-1(APLP1). Still further preferred is a method of contacting compositionscomprising APIP with amyloid precursor like protein-2 (APLP2). Suchmethods are useful, e.g. to inhibit the activity of the substrate suchas APP, APLP1, or APLP2, or to label the substrate, e.g. by labelingAPIP and using it to specifically bind to and thus allow thevisualization of the substrate or a cell or tissue expressing thesubstrate.

Another embodiment is directed at a method for reducing catabolism ofextracellular secreted amyloid beta precursor protein (APP) whichcomprises contacting a mammalian cell with APIP. Preferably the saidmammalian cell produces APP. The mammalian cell is preferably a neuronalcell. The mammal is preferably a rodent, canine, or primate.

Another embodiment is directed at a method for reducing catabolism ofextracellular secreted APLP 1 which comprises contacting a mammaliancell with APIP. Preferably the said mammalian cell produces APLP1. Themammalian cell is preferably a neuronal cell. The mammal is preferably arodent, canine, or primate.

Another embodiment is directed at a method for reducing catabolism ofextracellular secreted APLP2 which comprises contacting a mammalian cellwith APIP. Preferably the said mammalian cell produces APLP2. Themammalian cell is preferably a neuronal cell. The mammal is preferably arodent, canine, or primate.

Amyloid plaques in the brain contribute to disruption of neuronalconductivity which leads to disturbances in behavior, perception, memoryand mood. Another preferred embodiment of the invention is directed to amethod of preventing or alleviate mood disorders by contactingcompositions comprising APIP neuronal cells. Further preferred is amethod to prevent or alleviate schizophrenia by contacting compositionscomprising APIP with neuronal cells. Still further preferred is a methodto prevent or alleviate Alzheimer's disease by contacting compositionscomprising APIP with neuronal cells.

Amyloidosis also occurs in the pancreas and may contribute to thedevelopment of glucose intolerance, insulin insufficiency, or diabetes.A preferred embodiment is directed to a method of preventing ofalleviating glucose intolerance by contacting compositions comprisingAPIP with pancreatic cells. Further preferred is a method to prevent oralleviate insulin insufficiency by contacting compositions comprisingAPIP with pancreatic cells. Still further preferred is a method toprevent or alleviate diabetes by contacting compositions comprising APIPwith pancreatic cells.

It should be appreciated that preferred compositions of the invention tobe used in methods of the invention described for clone47-14-1-C3-CL0_(—)5 of SEQ ID NO:23 include polypeptides of SEQ ID NO:24(APIP), and fragments thereof, and compositions comprising thepolypeptides of SEQ ID NO:24, and fragments thereof.

Protein of SEQ ID NO:28 (Internal Designation Clone117401_(—)106-006-4-0-B11-F)

The cDNA of clone 117401_(—)106-006-4-0-B11-F (SEQ ID NO:27) encodes theprotein of SEQ ID NO:28. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:28 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone117401_(—)106-006-4-0-B11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:27described throughout the present application also pertain to the nucleicacids included in Clone 117401_(—)106-006-4-0-B11-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments. The gene for the proteinof SEQ ID NO:28 is located on chromosome 8.

The protein of SEQ ID NO:28 is referred to herein as Frangiopogen.Frangiopogen is highly expressed in human fetal liver and lung. Itstimulates liver regeneration, has mitogenic activity and is activelyinvolved in embryonic development. Frangiopogen is involved in complexregulatory processes including cell proliferation and angiogenesis.

In a preferred embodiment of the invention, Frangiopogen is used intissue treatment compositions to promote wound healing, preferably afterinjury, such as ischemia, or after surgery, including general surgery,ear-, nose- and throat surgery, tissue transplantation, dermal or dentalor artificial joint transplants, or plastic surgery. Further preferredare uses for Frangiopogen in tissue treatment compositions for tissueregeneration.

Preferred tissue treatment compositions of the present invention includephysiologically acceptable formulations comprising the protein of SEQ IDNO:28. Further preferred are physiologically acceptable formulationscomprising the protein of SEQ ID NO:28 in combination with an additionalcompound such as any or all of the compounds selected from the groupconsisting of fibrin, fibrinogen, thrombin, factor XIII, calciumchloride, a plasminogen activator, a plasmin inhibitor (such asaprotin), a growth factor, and a polysaccharide such as hyaluronic acid.Still further preferred are formulations comprising the protein of SEQID NO:28 alone or in compositions, e.g. as described in U.S. Pat. Nos.6,083,902 and 5,631,011, herein incorporated by reference in theirentireties.

In further embodiments, the tissue treatment compositions of theinvention are used in methods of treating injuries comprising the stepof contacting a wound or injured tissue with a healing or regenerativeeffective amount (an amount that would increase the rate or progressionof healing or regeneration as compared to the same wound or injuredtissue not treated with a composition of the present invention) of aFrangiopogen polypeptide. Further embodiments include use of the tissuetreatment compositions of the invention for topical application to asite of injury (e.g. as defined as a site in which the integument isdamaged in such a way as to expose the dermis), following an accident orfollowing surgery comprising the step of contacting the injured tissuewith a healing or regenerative effective amount of a Frangiopogenpolypeptide. In still further embodiments, the tissue treatmentcompositions of the invention, alone or in combination with chondrocytessuch as embryonic chondrocytes, are used in methods to treat jointcartilage and bone defect repair.

The present invention provides for methods of stimulating proliferationof endothelial cells comprising the step of contacting endothelial cellswith a proliferative effective amount of a Frangiopogen polypeptide ofthe present invention. Preferably the endothelial cells are vascularendothelial cells, arterial or venous. Further preferably, the methodresults in angiogenesis or the process of vascularization of a tissueinvolving the development of new capillary blood vessels. Preferably,angiogenesis occurs in a mammal, more preferably the mammal is a dog,cat, horse, cow, pig or human.

In addition, the present invention provides for an antibody thatspecifically binds a Frangiopogen polypeptide of the present invention.The antibody may be monoclonal or polyclonal. The invention alsoprovides for a method of inhibiting the growth of endothelial cellscomprising the step of contacting a biological sample comprisingendothelial cells with a growth inhibiting effective amount of ananti-Frangiopogen antibody. Preferably, the endothelial cells arevascular endothelial cells, arterial or venous. Further preferably, themethods results in the inhibition of angiogenesis or blood vesselgrowth. Further preferably, the inhibition of angiogenesis occurs in amammal, more preferably the mammal is a dog, cat, horse, cow, pig orhuman.

Alternatively, the invention provides for a Frangiopogenpolypeptide-cytotoxic agent conjugate, whereby the cytotoxic agent iscovalently or noncovalently, recombinantly or nonrecombinantly, attachedor conjugated to a Frangiopogen polypeptide using cytotoxic agents andmethods well known in the art. The invention also provides for a methodof inhibiting the growth of endothelial cells comprising the step ofcontacting a biological sample comprising endothelial cells or anindividual with a growth inhibiting or endothelial cell killingeffective amount of a Frangiopogen-cytotoxic agent conjugate.Preferably, the endothelial cells are vascular endothelial cells.Further preferably, the methods results in the inhibition ofangiogenesis or blood vessel growth. Further preferably, the inhibitionof angiogenesis occurs in a mammal, more preferably the mammal is a dog,cat, horse, cow, pig or human. To examine whether a particularanti-Frangiopogen antibody or a Frangiopogen-cytotoxic agent conjugateis useful to disrupt vascular growth or angiogenesis, models well knownin the art may be sued, e.g., the chick chorioallantoic membrane assay.

Preferred polypeptides for use in the methods of the present inventioninclude the polypeptides of SEQ ID NO:28 comprising the amino acidsequence:

MRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVVDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGXFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHXANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI; a polypeptidecomprising the amino acid sequence of:MAKVFSFILVTTALIMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDEDTVVDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI; a polypeptide comprising the amino acid sequenceof:SPISNCEITITDPGKFYNSNSVFSRGNMAKVFSFILVTTALXMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVVDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMK FSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWY SLKSVVMKIR PNDFIPNVI; a polypeptide comprisingthe amino acid sequence of:MAKVFSFILVTTALIMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVVDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFYVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI; and a polypeptide comprising the amino acidsequence of:MKLANWYWLSSAVLATYGFLVVANNETEEIKDERAKDVCPVRLESRGKCEEAGECPYQVSLPPLTIQLPKQFSRIEEVFKEVQNLKEIVNSLKKSCQDCKLQADDNGDPGRNGLLLPSTGAPGEVGDNRVRELESEVNKLSSELKNAKEEINVLHGRLEKLNLVNMNNIENYVDSKVANLTFVVNSLDGKCSKCPSQEQIQSRPVQHLIYKDCSDYYAIGKRSSETYRVTPDPKNSSFEVYCDMETMGGGWTVLQARLDGSTNFTRTWQDYKAGFGNLRREFWLGNDKIHLLTKSKEMILRIDLEDFNGVELYALYDQFYVANEFLKYRLHVGNYNGTAGDALRFNKHYNHDLKFFTTPDKDNDRYPSGNCGLYYSSGWWFDACLSANLNGKYYHQKYRGVRNGIFWGTWPGVSEAHPGGYKSSFKEAKMMIRPKHFKP. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.Proteins of SEQ ID NO:10 (Internal Designation Clone147103_(—)106-024-1-0-H6-F), SEQ ID NO:12 (Internal Designation Clone224168_(—)116-096-3-0-G11-F), SEQ ID NO:16 (Internal Designation Clone225432_(—)116-083-3-0-C6-F), and SEQ ID NO:14 (Internal DesignationClone 243303_(—)116-118-4-0-A3-F)

The polynucleotides of SEQ ID NOs:9, 11, 13 and 15 and the polypeptidesof SEQ ID NOs:10, 12, 14, and 16, respectively, encode the soluble Lowdensity lipoprotein receptor-Related Protein-10 (sLRP10)

MSASCCLSWCPAKAKSKCGPTFFPCASGIHCIIGRFRCNGFEDCPDGSDEENCTANPLLCSTARYHCKNGLCIDKSFICDGQNNCQDNSDEESCESSQAIFPQITVS. Preferred polynucleotidesand polypeptides of the invention comprise the nucleic acid sequences ofSEQ ID NOs:9, 11, 13, and 15 and amino acid sequences of SEQ ID NOs:10,12, 14, and 16. It will be appreciated that all characteristics and usesof the polynucleotides of SEQ ID NOs:9, 11, 13, and 15 and polypeptidesof SEQ ID NOs:10, 12, 14, and 16 described throughout the presentapplication also pertain to the human cDNAs of Clones147103_(—)106-024-1-0-H6-F, 224168_(—)116-096-3-0-G11-F,243303_(—)116-118-4-0-A3-F, and 225432_(—)116-083-3-0-C6-F, and thepolypeptides encoded thereby. Preferred compositions of the inventioninclude polynucleotides and polypeptides of Clones147103_(—)106-024-1-0-H6-F, 224168_(—)116-096-3-0-G11-F,243303_(—)116-118-4-0-A3-F, and 225432_(—)116-083-3-0-C6-F; SEQ IDNOs:9, 11, 13, and 15; SEQ ID NOs:10, 12, 14, and 16. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

sLRP10 is a non-membrane, soluble member of the Low Density LipoproteinReceptor (LDLR) family. This family is characterized by the presence ofa number of conserved, cysteine-rich LDLR domains. This domain folds toform a defined ligand-binding structure. Most members of the LDLR familyare transmembrane proteins that function in clathrin-mediatedendocytosis of various ligands. These ligands are usually then destroyedby lysosomal degradation. However, shorter, secreted family members havebeen described (U.S. Pat. No. 5,496,926 and Quinn, K. et al., Exp. CellRes. 251: 433-41(1999) which disclosures are hereby incorporated byreference in their entirety). The LDLR family of proteins is capable ofbinding a variety of protein and lipoprotein ligands. Furthermore,certain viruses target the LDLR domain to gain entry to cells expressingLDLR family members. LDLR proteins are expressed on a variety of celltypes including hepatocytes, neurons, fibroblasts, epithelial, adipose,muscle, and pancreatic cells.

High levels of Low Density Lipoprotein (LDL), Very Low DensityLipoprotein (VLDL), chylomicrons, and Apolipoprotein E (ApoE) areassociated with atherosclerosis and other cholesterol-associateddisorders. These molecules are subjects of intense study in the medicalfield. As a preferred embodiment, sLRP10 is used to bind LDL, VLDL,chylomicrons, and ApoE. While many members of the LDLR family, such asLDLR and alpha-2-macroglobulin receptor, are very large (>400 kD)membrane spanning proteins, sLRP10 is relatively small and not membraneassociated. Thus, sLRP10 is an easily purified polypeptide that can beused for binding LDLR domain ligands. As a part of this embodiment,sLRP10 polypeptide is covalently or non-covalently attached to a solidmatrix and allowed to bind LDL, VLDL, chylomicrons, or ApoE in solutionusing techniques well known in the art. Once bound, these proteins canbe purified using the following steps: i) wash the solid matrix to getrid of contaminants, ii) elute the protein of interest using morestringent conditions, e.g., increasing salt concentration.

Additional aspects of this embodiment include methods of detecting andquantifying LDL, VLDL, chylomicrons, or ApoE bound to sLRP10 usingtechniques common in the art (e.g., Western blotting, ELISA, or use of alabeled secondary detection method) comprising the steps of obtaining abiological sample suspected of containing LDL, VLDL, chylomicrons, orApoE; contacting said sample with an LDL, VLDL, chylomicrons, or ApoEbinding sLRP10 polypeptide of the present invention under conditionssuitable for binding of sLRP10 to LDL, VLDL, or ApoE; detecting thepresence or absence of LDL, VLDL, or ApoE by detecting the presence orabsence of sLRP10 bound to LDL, VLDL, or ApoE. This embodiment isuseful, for example, as a diagnostic tool for detecting plasma levels ofthese proteins.

In another embodiment of the invention, the sLRP10 polypeptide is usedto bind LDL, VLDL, chylomicrons, and ApoE in vivo and remove thesemolecules from the bloodstream. In this embodiment, the sLRP10polypeptide may further be expressed as a fusion protein with apolypeptide signal specifying excretion from the body. The invention isdelivered to individuals at risk of atherosclerosis or arteriallipoprotein deposits of LDL, VLDL, chylomicrons, or ApoE as determinedby common medical techniques including those described in U.S. Pat. No.5,652,224, incorporated herein by reference in its entirety, andcomprising the steps of i) determining the familial predisposition ofthe individual for these disorders, ii) obtaining a biological samplefrom the individual, and iii) subjecting that sample to analysis forlipoprotein content. Delivery includes administering an appropriateamount of sLRP10 polypeptide to the bloodstream of the diagnosedindividual, e.g., by injection.

ApoE is also associated with the pathogenesis of diabetes. Abnormallyhigh levels of ApoE are linked to amyloid plaques and destruction ofpancreatic P-cells. Furthermore, ApoE has antioxidant activity (Miyataand Smith, Nature Genet. 14: 55-61 (1996) which disclosures are herebyincorporated by reference in their entirety) and oxidative damagedestroys P-cells in type 1 diabetes (Bach J., Endocrin. Rev. 15: 516-542(1994) and PCT application WO9846743, incorporated herein by referencein its entirety). This embodiment of the invention could further bedelivered to patients suffering from or at risk of diabetes to reducelevels of pancreatic ApoE. In this embodiment, the sLRP10 polypeptidemay further be expressed as a fusion protein with a polypeptide signalspecifying excretion from the body. An appropriate dosage of sLRP10 maybe delivered specifically to the bloodstream, by injection for example,or to pancreatic cells using methods known in the art including thosedescribed in U.S. Pat. No. 5,652,224, incorporated herein by referencein its entirety. These include steps comprising i) construction of arecombinant viral vector comprising the DNA of, or corresponding to, aportion of the genome of an adenovirus, which portion is capable ofinfecting a pancreatic cell, operatively linked to the nucleotidesequence of the invention and the regulatory sequences directing itsexpression; ii) delivery of an effective amount of the recombinantadenoviral vector to an individual at risk for diabetes.

The polypeptide sLRP10 invention can bind ApoE as well as the amyloidprecursor protein (APP), both of which are associated with thepathogenesis of Alzheimer's disease (Kounnas, M. Z., et al., Cell82:331-40 (1995) which disclosures are hereby incorporated by referencein their entirety). As a further embodiment of the invention, sLRP10polypeptide is used to bind these proteins in neuronal cell populationsto allow study of Alzheimer's pathogenesis. In particular, the inventionis directly added to a population of neurons to block ApoE activity andstudy the formation of amyloid plaques.

sLRP10 is also able to bind the protooncogene Wnt-1 (Tamai, K., et al.,Nature 407:530-35 (2000) which disclosures are hereby incorporated byreference in their entirety). Wnt-1 usually functions as a solublegrowth factor that binds to Frizzled receptors but Wnt-1 has also beenassociated with transformation of cells (van Ooyen, A., Cell 39:233-40(1984) which disclosures are hereby incorporated by reference in theirentirety). Additionally, Wnt-1 has been associated with schizophrenia(Shackleford, G., et al., Neuron 11:865-75 (1993) which disclosures arehereby incorporated by reference in their entirety), making this proteinof particular interest to the biomedical community. Another embodimentof the sLRP10 polypeptide invention provides a method to study Wnt-1 andits effects using techniques common to the art. This embodiment providesa method of purifying Wnt-1 protein from a biological solution usingsteps comprising: i) attaching sLRP10 to a solid matrix; ii) applying asolution containing Wnt-1; iii) allowing Wnt-1 to bind to sLRP10; iv)washing and eluting Wnt-1. Purifying Wnt-1 is useful for a number ofapplications, for example to use purified Wnt-1 as a growth factor toadminister to cells, to generate antibodies against Wnt-1, and others.Additionally, this embodiment of the sLRP10 polypeptide is used to bindWnt-1 in solution and prevent its association with Frizzled receptors,thereby preventing molecular signaling events leading to cell growth,proliferation, and/or transformation.

sLRP10 binds to viruses comprising the Rous sarcoma, Flaviviridae(including hepatitis C), and Rhinovirus (including those responsible forthe “common cold”) families (Bates, P., et al., Cell 93:1043-51 (1993),Agnello, V., et al., PNAS 96:12766-71 (1999), Hofer, F., et al., PNAS91:183942 (1994) which disclosures are hereby incorporated by referencein their entirety). As a preferred embodiment of the invention, thesLRP10 polypeptide is used to bind viruses in solution. This embodimentcan be used to detect and quantify virus by techniques common to the art(e.g., fluorescent labeling of sLRP10) comprising steps of obtaining abiological sample suspected of containing virus from at least one of theRous sarcoma, Flaviviridae, or Rhinovirus families; contacting saidsample with labeled or otherwise detectable sLRP10 polypeptide; anddetecting and quantifying virus by visualizing the labeled sLRP10.

Membrane spanning LDLR family members are targeted by viruses of theRous sarcoma, Flaviviridae, and Rhinovirus families for entry intocells. However, as sLRP10 is not associated with the cellular membrane,it acts to block viral binding to LDLR proteins on the cells thatexpress these receptors, thereby preventing infection of those cells. Asa preferred embodiment of the invention, the sLRP10 protein is used tobind virus and prevent infection of LDLR family-expressing cells usingmethods known in the art including U.S. Pat. No. 5,496,926, incorporatedherein by reference in its entirety. This embodiment may be carried outby steps comprising: i) adding the sLPR10 polypeptide directly to cells,e.g. cells that express an LDLR family receptor, that may be exposed toa viral sample and ii) preventing the infection of said cells by virusesof the Rous sarcoma, Flaviviridae, and Rhinovirus families.

Protein of SEQ ID NO:20 (Internal Designation Clone158523_(—)106-030-2-0-A3-F)

The cDNA of Clone 158523_(—)106-030-2-0-A3-F (SEQ ID NO:19) encodes theOsteoAngioRemodeling (OAR) protein comprising the amino acid sequence

MRAWIFFLLCLAGRALAAPQQEALPDETEVVEETVAEVTEVSVGANPVQVEVGEFDDGAEETEEEVVAENPCQNHHCKHGKVCELDENNTPMCVCQDPTSCPAPIGEFEKVCSNDNKTFDSSCHFFATKCTLEGTKKGHKLHLDYIGPCKYIPPCLDSELTEFPLRMRDWLKNVLVTLYERDEDNNLLTEKQKLRVKKIHENEKRLEAGDHPVELLARDCQAVSARKAKIKSEM (SEQ ID NO:20).Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:20 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 158523_(—)106-030-2-0-A3-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:19 described throughout the present application alsopertain to the nucleic acids included in Clone158523_(—)106-030-2-0-A3-F. A preferred embodiment of the invention isdirected toward the compositions comprising SEQ ID NO:19, SEQ ID NO:20,or Clone 158523_(—)106-030-2-0-A3-F. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding the fragments. Another preferred embodiment ofthe invention is directed toward compositions comprising polypeptidefragments of at least six amino acids within SEQ ID NO:20:LLARDCQAVSARK, including those having a biological activity describedherein, and the corresponding polynucleotides. Preferred polypeptides ofthe present invention include polypeptide fragments of SEQ ID NO:20comprisingKKIHENEKRLEAGDHPVELLARDCQAVSARKAKIKSEM and the correspondingpolynucleotides. Further preferred polypeptides of the present inventioninclude polypeptide fragements of SEQ ID NO:20 comprisingDYIGPCKYIPPCLDSELTEFPLRMRDWLKNVLVTLYERDEDNNLLTEKQKLRVKKIHENEKRLEAGDHPVELLARDCQAVSARKAKIKSEM and the corresponding polynucleotides.Polypeptide fragments of SEQ ID NO:20 having a biological activity ofthose described herein and polynucleotides encoding the same are alsoincluded in the invention. Biological activities include increasing bonedensity when contacted with osteoblasts, tissue remodeling, and woundhealing.

The polypeptides of the OsteoAngioRemodeling (OAR) protein of SEQ IDNO:20 encode a carboxy-terminal variant of the human Osteonectin (alsoSPARC/BM-40) protein. OAR is encoded by the polynucleotides of SEQ IDNO:19 and represents an alternative splice variant of the full-lengthOsteonectin cDNA. This splice variant is characterized by the presenceof an alternative carboxy-terminal 15 amino acids starting at residue219 of the 303-amino acid Osteonectin protein.

OAR, like Osteonectin, is a non-collagenous, extracellularmatrix-associated protein. Expression is found in a number of cell typesthat include osteoblasts, platelets, and vascular epithelia, and isupregulated in sites of proliferation and extracellular matrix (ECM)remodeling. OAR is a modular protein whose domains mediate structure andprotein-protein interactions. OAR lacks domain IV of full-lengthOsteonectin, which contains one of two EF-hand motifs. OAR bindsmolecules such as collagen, PDGF, and FGF. Collagen type bindingspecificity is in part determined by differential N-glycosylation ofamino acids 71 and 99. This level of regulation is tissue-specific, sothat OAR from the bone binds collagens I, III, and V, yolk sac-derivedOAR binds only III and V, and platelet-derived OAR does not bindcollagen at all. Furthermore, binding decreases in low pH conditions.OAR plays a role in regulating cell mobility, proliferation, bone andtissue remodeling, and metalloproteinase production. OAR is involved inosteoporosis, osteoarthritis, atherosclerosis, angiogenesis, obesity,and metastatic tumors.

OAR is associated with increased bone density and remodeling. OAR isalso associated with metalloproteinase production, which is vital forbone remodeling. As a preferred embodiment, the OAR polypeptide of theinvention is used to increase the activity of osteoblasts using methodscommon to the art, for example, by adding a osteoblast-stimulatingamount of OAR to increase bone production to a culture of osteoblasticcells. This embodiment is applied to increase the productivity ofosteoblasts for purposes comprising study or replacement therapy. As afurther embodiment, OAR is used in methods of bone remodeling such asthose described in Gerber, H., et al. (1999) Nat. Med. 5:623-8, whichdisclosures are hereby incorporated by reference in their entirety. Forexample, OAR is used in a method to promote osteoblast differentiationand bone remodeling by inducing metalloproteinase or osteocalcinproduction by contacting OAR with osteoblastic cells in culture.Furthermore, OAR is used in a method to promote in vivo osteoblastdifferentiation by contacting OAR with an area of potential bone growth,for instance, in the growth plate of the femur or in the hip which isoften the site of fracture. An effective amount of OAR is delivered tothe site by injection or other methods common to the art andeffectiveness determined using any suitable method such as X-rays, ormethods described in Delany, A., et al. (2000) J. Clin. Invest. 105:915-23, which disclosure is hereby incorporated by reference in itsentirety.

Cells derived from certain tissues adhere to specific collagens. OARbinds collagen types I, III, and V which are found, for example, inepithelia and bone tissue. This allows OAR to act as an anti-adhesionfactor by inhibiting normal interaction of collagen in the ECM to cellsurface adhesion molecules. This activity is associated with cellmigration and differentiation. Furthermore, OAR is associated withincreased metalloproteinase expression, which leads to ECM degradationand tissue remodeling. Thus, a preferred embodiment of the invention isdirected to a method of using OAR in tissue remodeling, wherebycontacting OAR with osteoblasts to inhibit binding of collagen to cellsallows tissue remodeling. Further preferred is a method to use OAR inwound healing (e.g., from surgical damage or chronic conditions such asdiabetic ulcers), tissue grafts, necrotic or hypoxic tissue in ECMenvironments comprising collagen types I, III, and V that bind OAR. Amethod to treat these conditions includes steps comprising: i)identifying the ECM of the tissue in need of repair as one that bindsOAR using methods common in the art (e.g., applyingfluorescently-labeled OAR to an ECM sample and visualizing bymicroscopy); ii) localizing an effective amount of OAR to the wound areaeither directly or by injection; iii) allowing ECM remodeling to occuras OAR inhibits cell adhesion.

Osteonectin binds to VEGF, which regulates blood vessel formation. Thisinteraction prevents VEGF binding to its receptor. The OAR polypeptidelacks a VEGF-binding domain while it retains its ability to bind the ECMand affect remodeling (Kupprion, C., et al. (1998) J. Biol. Chem.273:29635-40 which disclosure is hereby incorporated by reference in itsentirety). In a preferred embodiment of the invention, OAR polypeptideis used to replace Osteonectin in conditions that require VEGF activityin addition to the ECM interactions that mediate wound healing andtissue remodeling. This is accomplished in steps comprising: i)obtaining a cell or tissue sample in culture that contains at least VEGFand VEGF-responsive cells; ii) adding OAR to the culture in an amounteffective for ECM binding, iii) allowing OAR to enable ECM remodeling aswell as VEGF signaling to aid in angiogenesis and tissue healing. Inaddition, expression of Osteonectin may be inhibited by introducing IL-1to the affected area and as described in Nakamura, S., et al. (1996)Arthritis Rheum. 39:539-51, which disclosure is hereby incorporated byreference in its entirety. As a further embodiment, the invention isapplied to the growth and healing of necrotic or hypoxic tissue, tissuegrafts, and bone-associated tissue. The OAR polypeptide is delivered tothese tissues using methods common to the art such as injection or useof OAR polypeptide fused to a targeting molecule specific for the tissueof interest.

In the extreme, decreased “contact inhibition” from the ECM to the cellsurface is linked to tumor formation and metastasis. As OAR inhibitscontact of cells to specific types of collagen in the ECM, OAR isinvolved in metastasis of a number of tumor cell types including breastand prostate carcinomas. In a preferred embodiment of the invention, theOAR polypeptide is used to develop inhibitors of its collagen-bindingactivity to prevent ECM invasion. This invasion includes theproliferation of cells into inappropriate tissues, such as that observedin rheumatoid arthritis and cancers including breast and prostatecarcinomas. Inhibitors of OAR are comprised of antibodies raised againstthe carboxy-terminal 15 amino acids of the OAR polypeptide and smallmolecules that interfere with OAR collagen binding activity. OAR bindingto ECM environments is determined using methods common to the art suchas applying fluorescently-labeled OAR to a tissue sample and visualizingby microscopy. Effectiveness of OAR inhibitors is determined using theaforementioned method or by observing cell invasion of the ECM asdescribed by Kato, Y., et al. (1998-99) Invasion Metastasis 18:105-147,which disclosure is hereby incorporated by reference in its entirety. Anexample use of this embodiment would include methods comprising thesteps: i) purifying the OAR inhibitor such as an antibody using methodscommon in the art (e.g.—affinity chromatography); ii) determining a siteof inappropriate ECM invasion using methods common to the art such astissue imaging, X-ray, or palpation; iii) localizing an effective amountof OAR inhibitor to the site to allow cell surface-collagen interactionsand prevent ECM invasion. Localization of the OAR inhibitor is effectedusing methods common in the art such as injection. Further included inthe invention is a method for delivering the OAR polypeptide fused to atargeting molecule specific for the tissue of interest.

OAR binds to growth factors including PDGF, which can induce cellmigration and proliferation, and inhibits binding of the growth factorto its receptor under certain conditions. As a preferred embodiment ofthe invention, the OAR polypeptide is used to inhibit signaling throughgrowth factor receptors such as the PDGF receptor. This embodiment isuseful in preventing inappropriate growth of PDGF-responsive cells, suchas dermal fibroblasts (e.g., in the case of hypertrophic scars) andplatelets (e.g., in cases of malignant lymphomas). This embodiment iscarried out, for instance to reduce the volume of a hypertrophic scar,by identifying a region with excess scar tissue using methods describedby Nedelec, B., et al. (2000) J. Burn Care Rehabil. 21:205-12, whichdisclosure is hereby incorporated by reference in its entirety;administering an effective amount of OAR to the scar directly or byinjection; and monitoring the scar using aforementioned method or otherscommon to the art.

Protein of SEQ ID NO:30 (Internal Designation Clone133431_(—)105-092-4-0-G11-F)

The cDNA of clone 133431_(—)105-092-4-0-G11-F (SEQ ID NO:29) encodes avariant of the ALEX-1 protein with the amino acid sequence

MGRTREAGCVAAGVVIGAGACYCVYRLAWGRDENEKIWDEDEESTDTSXIGVETVKGAKTNAGAGSGAKLQGDSEVKPEVSLGLEDCPGVKEKAHSGSHSGGGLEAKAKALFNTLKEQASAKAGKGARVGTISGNRTLAPSLPCPGGRGGGCHPTRSGSRAGGRASGKSKGKARSKSTRAPATTWPVRRGKFNFPYKIDDILSAPDLQKVLNILERTNDPFIQEVALVTLGNNAAYSFNQNAIRELGGVPIIAKKKKK (SEQ ID NO:30). It will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:30 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone133431_(—)105-092-4-0-G11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:29described throughout the present application also pertain to the nucleicacids included in Clone 133431_(—)105-092-4-0-G11-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:29, SEQ ID NO:30, and Clone 133431_(—)105-092-4-0-G11-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments. Thegene of SEQ ID NO:29 is located on the X-chromosome. It encodes a newarmadillo repeat protein with a death effector domain and is involved incell-cell adhesion, cell signaling and apoptotic processes and is herebyreferred to as Armapoptin.

Armapoptin promotes cell growth and differentiation during embryonicdevelopment. It is part of multi-protein complexes, which mediatecell-cell adhesion, anchorage to the actin cytoskeleton with adjacentcells, and a signal in response to cell adhesion to initiate cellpolarity and the formation of epithelia. Armapoptin complexes, whichinclude E-cadherin and different cadherin-binding proteins includingβ-catenin can also be associated with a tumor suppressor protein such asAdenomatous Polyposis Coli (APC), which is mutated in hereditary coloncancer. Cell-cell adhesion in normal differentiation processes andmalignant proliferation is mediated by the armadillo domain serving as ascaffold for the assembly of multi-protein complexes.

The N-terminal region of Armapoptin contains a death effector domain(DED) comprising residues RLAWGRDENEKIWDEDEES. Death effector domainsare involved in caspase-dependent apoptotic processes. Armapoptin isexpressed in most tissues, but is not expressed or significantlyunderexpressed in breast carcinoma biopsies of patients as well as inepithelial based-tumor cell lines including ovarian carcinoma, cervixadenocarcinoma cells, lung carcinomas, and immortalized endothelial celllines such as t-HUE2.

In an embodiment, Armapoptin polynucleotides are used in a method ofgene therapies to restore cell-cell adhesion and to promotecaspase-dependent apoptosis, preferably in epithelial cell-based tumorsincluding breast carcinoma, ovarian carcinoma, lung carcinoma, non-smallcell lung carcinoma (NSCLC), and squamous cell carcinoma of head andneck (SCCHN). Preferred compositions of Armapoptin to be used in methodsof gene therapy, further referred to as “gene therapy compositions ofArmapoptin” are compositions comprising the full-length DNA, SEQ IDNO:29, or fragments thereof, encoding a polypeptide or fragmentsthereof, including the sequences

aatcctagtcttcgtttggtccggttgcactcttcctatagcccagagggcgagagggcctgtggcctgggggaaggaggacgaggttctgcctggatcccagcaggacgctgtgccatttgggaacaaaggaatagtctgcctggaatccctgcagatcttggggccggaggccagtccaacccttggagcaggaagaaacgcaaagttgtcaagaaccaagtcgagctgcctcagagccggcccgcagtagctgcagactccgcccgcgacgtgtgcgcgcttctctgggccagagcgagcctgttttgtgctcgggttaagagatttgtcccagctataccgcgtggccgctggtgtggttatcggggctggtgcctgctactgtgtatacagactggcttggggaagagacgagaacgagaaaatctgggacgaagacgaggagtctacggacacctcakagattggggttgagactgtgaaaggagctaaaactaacgctggggcagggtctggggccaaacttcagggtgattcagaggtcaagcctgaggtgagtttgggactcgaggattgtccgggtgtaaaagagaaggcccattcaggatcccacagcggaggtggcctagaggccaaggccaaggcccttttcaacacgctgaaggaacaggcaagtgcaaaggcaggcaaaggggctagggtgggtaccatctctgggaacaggaccttgcaccgagtttaccctgcccaggaggcaggggtggaggctgccaccccaccaggagtggatctagggccgggggcagggcaagtggaaaatccaagggaaaggcccgaagtaagagcaccagggctccagctacaacatggcctgtccggagaggcaagttcaactttccttataaaattgatgatattctgagtgctcccgacctccaaaaggtcctcaacatcctggagcgaacaaatgatccttttattcaagaagtagccttggtcactctgggtaacaatgcagcatattcatttaaccagaatgccatacgtgaattgggtggtgtcccaattattgcaaaaaaaaaaaaaaaa,ortctgagtacc agctccccac tgccctgagg gcgggccggc ctgcggcggagggaaaaaggaagaggagaa ggaaattgtc ccgaatccct gcagtgggtc caagcctctcccgggtggccagtctttctg taggttgcgg cacaacgcca ggcaaaagaa gaggaaggaatttaatcctaatcggtggag gtcgattga gggtctgctg tagcaggtgg ctccgcttgaagcgagggaggaagttcct ccgatcagta gagattggaa agattgttgggagtggcacaccactagggaaaagaagaag gggcgaactg cttgtcttga ggaggtcaacccccacaatc agctcttgtggccttgaagt ggctgaagac gatcaccctc cacaggcttgagcccagtcc cacagccttcccccagc ctgagtgact actctattcc ttggtccctg ctattgtcggggacgttgcatgggctacg ccaggaaagt aggctgggtg accgcaggcc tggtgattggggctggcgcctgctattgca tttatagact gactagggga agaaaacaga acaaggaaaaaatggctgagggtggatctg gggatgtgga tgatgctggg gactgttctg gggccaggtataatgactggtctgatgatg atgatgacag caatgagagc aagagtatag tatggtacccaccttgggctcggattggga ctgaagctgg aaccagagct agggccaggg caagggccagggctacccgggcacgtcggg ctgtccagaa acgggcttcc cccaattcag atgataccgttttgtcccctcaagagctac aaaaggttct ttgcttggtt gagatgtctg aaaagccttatattcttgaagcagctttaa ttgctctggg taacaatgct gcttatgcat ttaacagagatattattcgtgatctgggtg gtctcccaat tgtcgcaaag attctcaata ctcgggatcccatagttaaggaaaaggctt taattgtcct gaataacttg agtgtgaatg ctgaaaatcagcgcaggcttaaagtataca tgaatcaagt gtgtgatgac acaatcactt ctcgcttgaactcatctgt gcagcttg ctggactgagatt gcttacaaat atgactgtta ctaatgagtatcagcacatgcttgctaatt ccatttctga cttttttcgt ttattttcag cgggaaatgaagaaaccaaacttcaggttc tgaaactcct tttgaatttg gctgaaaatc cagccatgactagggaactgctcagggccc aagtaccatc ttcactgggc tccctcttta ataagaaagagaacaaagaagttattctta aacttctggt catatttgag aacataaatg ataatttcaaatgggaagaaaatgaaccta ctcagaatca attcggtgaa ggttcacttt ttttctttttaaaagaatttcaagtgtgtg ctgataaggt tctgggaata gaaagtcacc atgattttttggtgaaagtaaaagttggaa aattcatggc caaacttgct gaacatatgt tcccaaagagccaggaataacaccttgatt ttgtaattta gaagcaacac acattgtaaa ctattcattttctccaccttgtttatatgg taaaggaatc ctttcagctg ccagttttga ataatgaatatcatattgtatcatcaatgc tgatatttaa ctgagttggt ctttaggttt aagatggataaatgaatatcactacttgtt ctgaaaacat gtttgttgct ttttatctcg ctgcctagattgaaatattttgctatttct tctgcataag tgacagtgaa ccaattcatc atgagtaagctcccttctgtcattttcatt gatttaattt gtgtatcatc aataaaattg tatgttaatgctggaagggaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa.

Further preferred are compositions comprising PCR-based subcloning ofthe gene therapy compositions of Armapoptin into plasmid vectors such aspCMVβ or pSVβ, tissue-specific promoter-containing plasmids such as theMUC1 promoter, which allows epithelial cell specific expression and isup-regulated during malignancy, and the P450 arom promoter II for breastcarcinomas employing liposomal delivery systems by methods described inPatel, U.S. Pat. No. 6,225,090, 2001, Thierry, U.S. Pat. No. 6,110,490,2000; Wolff, et al., U.S. Pat. No. 6,228,844, 2001, Graham, et al., Int.J. Cancer 92:382-387, 2001, Zhou, et al, Cancer Res. 61:2328-2334, 2001,which disclosures are hereby incorporated in their entireties. Furtherpreferred are compositions comprising polynucleotides of the inventioncloned into adenoviral vectors (Beach, et al., U.S. Pat. No. 5,968,821,1999, and 6,211,334,2001; Mehtali, et al., U.S. Pat. No. 6,204,060,2001), and MoMLV-based retroviral vectors for gene delivery intodividing cells, i.e. tumor tissues according to methods described byHolt, et al., U.S. Pat. No. 6,177,410, 2001, which disclosures arehereby incorporated in their entirety.

Methods to deliver preferred compositions of Armapoptin polynucleotidesand fragments thereof, comprise local injection of preferredcompositions of the invention into tumor tissue or surrounding vessels,or ex vivo therapy. Further methods comprise tumor tissue specifictargeting of Armapoptin polynucleotides or fragments thereof in aplasmid via antibodies or other ligands, which recognize tumor-specificreceptors. These ligands will be covalently linked to polycations suchas poly-L-lysine or liposomes, and complexed with preferred gene therapycompositions of Armapoptin. Preferred tumor cell types to be used inmethods of gene therapy include breast carcinoma, cervix adenocarcinoma,ovarian carcinoma, lung carcinoma, and squamous cell carcinoma of headand neck derived from mammalian cells including rodent and human.Assessment of therapeutic efficacies will include tumor regressionfollowing delivery of preferred gene therapy compositions of Armapoptinas monitored by measurement of tumor circumference. Apoptosis will bemeasured by morphological assessments including retraction ofcytoplasmic extension, cell rounding and detachment, and via MTT assays,which measure mitochondrial function for viability, cell death andcaspase activity, and DNA fragmentation analysis as described byNoteborm, et al. U.S. Pat. No. 5,981,502, 1999; Boone, et al., J. Biol.Chem. 275:37596-37603, 2000; Shibata, et al., Cancer Gene Therapy.8:23-35, 2001; Lacour, et al., Cancer Research 61:1645-1651, 2001),which disclosures are hereby incorporated by reference in theirentireties.

Further embodiments include putative death effector domains fortherapeutic use in caspase-dependent cell death including incubation ofcarcinoma cells with compositions comprising polypeptides of preferredsequences comprising RLAWGRDENEKIWDEDEES and FADD DED-related domains asdescribed in Eberstadt, et al., Nature. 392:941-945, 1998, and Hackam,et al., J. Biol. Chem. 275:41299-41308, 2000, which disclosures arehereby incorporated by reference in their entireties, with the consensussequence

SSYRVLLLLISEELDSEELEVLLFLCNDDIPKRKLEIKTALDLFSALEEQGLLSEDNLSLLAELLYRLRRLDLLRRLFG.

Further, these DED domain-encoding sequences will be subcloned intoexpression vectors and used for cell transfections and apoptosis studiesas described above.

In another embodiment, Armapoptin polypeptides or fragments thereof willbe used as immunotherapeutics by covalent or noncovalent linkage to acell-specific (e.g. tumor cell-specific) antibody, or to a ligand whichis recognized by a tumor cell-specific receptor and internalized.Receptors which are abundantly expressed on tumor cells but not onintact, quiescent tissues to be employed in the present inventioninclude H11 [(C-antigen); Dan, et al., U.S. Pat. No. 6,207,153, 2001],tyrosine growth factor receptors including erbB-2 (HER-2-neu) (Suzuki,et al., Biochim Biophys Acta. 1525:191-196, 2001; Kumar, et al., SeminOncol. 27:84-91, 2000; Lango, et al., Current Opin Oncol. 13:168-175,2001), the folate receptor (Ward, Current Opin Mol Ther. 2:182-187,2000), human epidermal growth factor receptor (Schlessinger, et al.,U.S. Pat. No. 6,217,866, 2001), and endoglin on endothelial cells fortumor vascular targeting (Seon, U.S. Pat. No. 6,200,566, 2001), whichdisclosures are hereby incorporated by reference in their entirety.

The death effector domain causes neuronal cell death in Huntington'sdisease (Hackam, et al., J. Biol. Chem. 275:41299-41308, 2000, whichdisclosures are hereby incorporated by reference in their entirety) bystronger association with the mutant, glutamine rich protein, whichcauses the disease as opposed to wild-type huntingtin in healthyindividuals. Another embodiment uses Armapoptin and ALEX-1, partialsequences thereof including the death effector domainRLAWGRDENEKIWDEDEES, and the death effector domain of thehuntingtin-interacting protein (HIP-1), conserved among relatedsequences with the consensus peptide

SSYRVLLLLISEELDSEELEVLLFLCNDDIPKRKLEIKTALDLFSALEEQGLLSEDNLSLLAELLYRLRRLDLLRRLFG for competitive binding studies with wild-typehuntingtin and the disease-causing mutant. By contacting polypeptides ofthe invention with wt- and mt- (glutamine-rich) huntingtin,peptide-protein interactions will be analyzed by biophysical methods andvalidated using the following steps as described in Scalley, et al.,Biochemistry. 38:15927-15935, 1999; Chaillan-Huntington et al., J BiolChem. 275:5874-5879, 2000; Lohner et al., Biochim Biophys Acta.1462:141-156, 1999; Eberstadt, et al., Nature 392:941-945, 1998, whichdisclosures are hereby incorporated in their entireties.

Structural transitions in the denatured state ensemble, fluorescenceenergy transfer, and determination of peptide conformation andstructural characteristics using circular dichroism Isothermal titrationcalorimetry, fluorescence binding assays, and differential scanningcalorimetry to determine comparative K_(d) values, and strength ofinteractions.

Structure determination of polypeptide/huntingtin complexes by NMR andX-ray crystallography. Co-incubation of cell lines like 293 T cells withprotein-peptide complexes, and co-transfection of cells with wt- andmt-huntingtin-encoding plasmids and cloned oligonucleotides forcytotoxicity assays as well known in the art.

Another embodiment includes the method to use armadillo repeats ofarmapoptin, including

NFPYKIDDILSAPDLQKVLNILERTNDPFIQEVALVTLGNNAA, and

YSFNQNAIRELGGVPIIAKLIKTKDPIIREKTYNALNNLSV as single repeats, andnaturally occurring tandem array repeats

NFPYKIDDILSAPDLQKVLNILERTNDPFIQEVALVTLGNNAAYSFNQNAIRELGGVPIIAKLIKTKDPIIREKTYNALNNLSV for the restoration of cell-cell adhesion intreatment or prevention of cancer or other diseases or disorders whererestoration of cell-cell adhesion is sought, wherein said methodincludes contacting cells in need of cell-cell adhesion with eithermonomers or concatamerized forms, either recombinantly ornonrecombinantly, such as dimmers, trimers, or longer repeats, in acell-cell adhesion restorative amount of an Armapoptin polypeptide ofthe present invention.Protein of SEQ ID NO:26 (Internal Designation Clone545542_(—)182-1-2-0-D12-F)

The cDNA of clone 545542_(—)182-1-2-0-D12-F (SEQ ID NO:25) encodes the251 amino acid human Fibroblast Growth Factor-22 protein (FGF-22)comprising the amino acid sequence:

MLGARLRLWVCALCSVCSMSVLRAYPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGP EGCRPFAKFI(SEQ ID NO:26). Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:26 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone545542_(—)182-1-2-0-D12-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:25described throughout the present application also pertain to the nucleicacids included in Clone 545542_(—)182-1-2-0-D 12-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:25, SEQ ID NO:26, and Clone 545542_(—)182-1-2-0-D12-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

FGFs exert their biological effects through interaction with cognatesingle transmembrane, heparin-binding, fibroblast growth factorreceptors (FGFR) with intrinsic kinase activity, designated fibroblastgrowth factor receptor 1 (FGFR-1), fibroblast growth factor receptor 2(FGFR-2), fibroblast growth factor receptor 3 (FGFR-3) and fibroblastgrowth factor receptor 4 (FGFR-4). Physiologically, FGFs bind heparinsulfate proteoglycans which are sulfated glycosaminoglycans covalentlybound to core protein. The ability to bind heparin-like moietiesincludes FGFs within the more encompassing Heparin Binding Growth Factor(HBGF) superfamily of peptide growth factors. Additionally, FGFs bindthe cysteine-rich FGF-R (CFR), an integral single transmembrane proteinin a mutually exclusive manner with respect to the other FGFRs.

FGF-22 exibits a pattern of temporal and spatial expression in theembryonic and adult organism most pronounced in the brain, including butnot limited to the ventrolateral thalamic nucleus and thalamus. FGF-22is directly associated with the inherited disorder Autosomal DominantHypophosphatemic Rickets (ADHR), represented by missense mutations inFGF-22 polypeptide residues ARG176GLN and ARG179TRP of SEQ ID 26,respectively, resulting from FGF-22 nucleotide transitions at positionG527A and C535T, respectively of SEQ ID 25.

Included as an embodiment of the present invention is a method ofelevating serum phosphate levels to within physiologically acceptableconcentrations comprising the step of contacting kidney tissue or cells,in vitro or in vivo, with an effective amount of a FGF-22 polypeptide.The polypeptide of the present invention may be employed in combinationwith a suitable physiologically acceptable carrier to comprise aphysiologically acceptable composition for administration. Suchcompositions comprise a therapeutically effective amount of the FGF-22polypeptide and a physiologically acceptable carrier or excipient. Sucha carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Preferably, thekidney cells are nephron renal tubules and associated vascularcomponents (collectively designated the glomerular capsule) capable ofaltering tubular reabsorption, and/or distal or collecting tubules.Preferably, the kidney tissue or cell is contacted by administering aFGF-22 polypeptide to an individual. As used herein, the term“individual” includes members of the animal kingdom including but notlimited to human beings. Preferably, the FGF-22 polypeptide isadministered parenterally, more preferably intraperitoneal.

Further included in the present invention is a method of attenuatingosteomalacia or tumor-induced osteomalacia comprising contacting osseoustissue (osteocytes, osteoblasts, osteoclasts) with an osteomalaciainhibiting effective amount of a FGF-22 polypeptide. The polypeptide ofthe present invention may be employed in combination with a suitablephysiologically acceptable carrier to comprise a physiologicallyacceptable composition for administration. Such compositions comprise atherapeutically effective amount of the FGF-22 polypeptide and aphysiologically acceptable carrier or excipient. Such a carrier includesbut is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration. Preferably, the osseus tissue or cell iscontacted by administering a FGF-22 polypeptide to an individual.Preferably, the FGF-22 polypeptide is administered parenterally, morepreferably intraperitoneal.

In another embodiment of the present invention is a method ofattenuating osteopenia comprising contacting osseus tissue (osteocytes,osteoblasts, osteoclasts) with an osteopenia inhibiting effective amountof a FGF-22 polypeptide. The polypeptide of the present invention may beemployed in combination with a suitable physiologically acceptablecarrier to comprise a physiologically acceptable composition foradministration. Such compositions comprise a therapeutically effectiveamount of the FGF-22 polypeptide and a physiologically acceptablelyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration. Preferably, the osseous tissue is contacted byadministering a FGF-22 polypeptide to an individual. Preferably, theFGF-22 polypeptide is administered parenterally, more preferablyintraperitoneal.

In another embodiment of the present invention is a method ofattenuating osseous bone matrix deposition, including defects associatedwith congenital malformations, osteogenesis imperfecta (types I-IV),osteoporosis (type I and/or type II), rickets, fracture remodeling,surgical repair and restoration, and associated with deficiencies inosteoid mineralization or deposition, comprising contacting osseoustissue (osteocytes, osteoblasts, osteoclasts) with a osteoid depositionor osteoid mineralization stimulating effective amount of a FGF-22polypeptide. The polypeptide of the present invention may be employed incombination with a suitable physiologically acceptable carrier tocomprise a physiologically acceptable composition for administration.Such compositions comprise a therapeutically effective amount of theFGF-22 polypeptide and a physiologically acceptablely acceptable carrieror excipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.Preferably, the osseous tissue is contacted by administering a FGF-22polypeptide to an individual. Preferably, the FGF-22 polypeptide isadministered parenterally, more preferably intraperitoneal.

In another embodiment of the present invention is a method ofattenuating bone resorption or jaw atropy associated with dental abscess(periapical or periodontal) formation or progression, congenital orderived edentulous conditions, or consequent to elective dentalextraction, comprising contacting oral cavity osseous tissue(osteocytes, osteoblasts, osteoclasts) of the mandible or maxilla,preferably located adjacent to the sulcular groove region, with aneffective amount of an FGF-22 polypeptide. The polypeptide of thepresent invention may be employed in combination with a suitablephysiologically acceptable carrier to comprise a physiologicallyacceptable composition for administration. Such compositions comprise atherapeutically effective amount of the FGF-22 polypeptide and aphysiologically acceptablely acceptable carrier or excipient. Such acarrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Preferably, theosseous tissue is contacted by administering a FGF-22 polypeptide to anindividual. Preferably, the FGF-22 polypeptide is administeredparenterally, by any convenient manner, typically by syringe or catheterat the location of targeted osteosynthesis. In a further embodiment ofthis invention is a method of facilitating osseointegration of dentalimplant prostheses comprising contacting oral cavity osseous tissue(osteocytes, osteoblasts, osteoclasts) of the maxilla and/or mandible aswell as osseous tissue i.e. autogeneic or allogeneic bone graft, ordental biomaterial matrix i.e. coral or hydroxyapatite, incorporatedwithin the dental implant device, or bioabsorbable cement inperi-implant region with an effective amount of an FGF-22 polypeptide.Following tooth extraction, implant osteotomies were prepared and FGF-22polypeptide included with a bioabsorbable cement placedcircumferentially within the osteotomies. Implant prostheses were placedinto the prepared sites including the FGF-22 dental cement (Meraw etal., (J Periodontol 71: 8-13, 2000)). Preferably, the osseous tissue iscontacted by administering a FGF-22 polypeptide to an individual. Thepolypeptide of the present invention may be employed in combination witha suitable physiologically acceptable carrier to comprise aphysiologically acceptable composition for administration. Suchcompositions comprise a therapeutically effective amount of the FGF-22polypeptide and a physiologically acceptable carrier or excipient. Sucha carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Preferably, theFGF-22 polypeptide is administered parenterally, by any convenientmanner, typically by syringe or catheter at the location of targetedosteosynthesis. FGF-22 polypeptide is alternatively or additionallyadministered directly associated with the biodegradable matrix of thedental implant using methods of Illi (U.S. Pat. No. 6,214,008/PCTWO98/46289), Gayer and Comfort (U.S. Pat. No. 6,214,049), and/orassociated with the bioabsorbable cement using the methods of Meraw, etal. (J Periodontol 71: 8-13, 2000), which disclosures are herebyincorporated by reference in their entireties.

A further embodiment of the current invention is a method offacilitating osteosynthesis of bone to attenuate acetablular erosion orosteonecrosis of the femoral head in advance of orthopedicosseointegration of hip joint implant prostheses for hip arthroplastycomprising contacting implant localized osseous tissue (osteocytes,osteoblasts, osteoclasts) of the hip joint, preferably the acetabularregion and/or femoral head, with a stimulating effective amount of aFGF-22 polypeptide. The polypeptide of the present invention may beemployed in combination with a suitable physiologically acceptablecarrier to comprise a physiologically acceptable composition foradministration. Such compositions comprise a therapeutically effectiveamount of the FGF-22 polypeptide and a physiologically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration. Preferably, the osseous tissue is contacted byadministering a FGF-22 polypeptide to an individual. Preferably, theFGF-22 polypeptide is administered parenterally, preferablyintraperitoneal, or by any convenient manner, or by syringe or catheterat the location of targeted osteosynthesis. FGF-22 polypeptide isadditionally administered by incorporation with the biodegradable matrixof the prosthetic joint implant.

An additional embodiment of this invention is a method of facilitatingosteosynthesis of bone to attenuate articular surface erosion orosteonecrosis of the femur and/or tibia and/or patella in advance oforthopedic osseointegration of knee joint implant prostheses for kneejoint arthroplasty, or osteochondral fracture repair, or the placementof orthopedic pins or screws, comprising contacting implant localizedosseous tissue (osteocytes, osteoblasts, osteoclasts) of the knee joint,preferably the articular surfaces, with a stimulating effective amountof a FGF-22 polypeptide. The polypeptide of the present invention may beemployed in combination with a suitable physiologically acceptablecarrier to comprise a physiologically acceptable composition foradministration. Such compositions comprise a therapeutically effectiveamount of the FGF-22 polypeptide and a physiologically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration. Preferably, the osseous tissue is contacted byadministering a FGF-22 polypeptide to an individual. Preferably, theFGF-22 polypeptide is administered parenterally, preferablyintraperitoneal, or by any convenient manner, typically by syringe orcatheter at the location of targeted osteosynthesis. FGF-22 polypeptideis additionally administered by incorporation with the biodegradablematrix of the prosthetic joint implant. FGF-22 is a potent inducer ofepithelial cell proliferation. Therefore, another embodiment of thisinvention is a method of stimulating epithelial cell proliferation orincreasing epithelial cell viability by contacting said cells, in vitroor in vivo, with a proliferative stimulating or viability increasingeffective amount of a FGF-22 polypeptide. More specifically a method ofpromoting wound repair or tissue healing, such as resultant from burn,ulcer (e.g., venous ulcers in diabetics), aging, post-operative damage,disease, or other insult, by stimulating epithelial cell proliferationor increasing epithelial cell viability by contacting said cells ortissue, in vitro or in vivo, with a proliferation stimulating orviability increasing effective amount of a FGF-22 polypeptide. Thepolypeptide of the present invention may be employed in combination witha suitable physiologically acceptable carrier to comprise aphysiologically acceptable composition for administration. Suchcompositions comprise a therapeutically effective amount of the FGF-22polypeptide and a physiologically acceptable carrier or excipient. Sucha carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Preferably, theepithelial tissue is contacted by administering a FGF-22 polypeptide toan individual. Preferably, the FGF-22 polypeptide is administeredparenterally, preferably intraperitoneal, or by any convenient manner,typically by syringe or catheter directly at the location of targetedepithelial proliferation.

FGF-22 is a potent regulator of connective tissue proliferation,including embryonic mesechymal cells, fibrobastic cells of areolar,collagenous and elastic connective tissue, chondrocytes of cartilage andosteocytes of bone. Therefore, another embodiment of this invention is amethod of stimulating fibroblast cell proliferation or increasingfibroblast cell viability by contacting said cells, in vitro or in vivo,with a proliferative stimulating or viability increasing effectiveamount of a FGF-22 polypeptide. A further specified embodiment of thepresent invention is a method of promoting wound repair or tissuehealing, in vitro and in vivo, such as resultant from bum, ulcer, aging,post-operative damage such as tendon and ligament repair (Chan, et al.,Acta Orthop Scand 71: 513-518, 2000; Kuroda, et al., Knee Surg SportsTraumatol Arthrosc 8:120-126, 2000), disease, or other insult, bystimulating connective tissue cell proliferation or increasingconnective tissue cell viability by contacting said cells, in vitro orin vivo, with a proliferation stimulating or viability increasingeffective amount of a FGF-22 polypeptide. The polypeptide of the presentinvention may be employed in combination with a suitable physiologicallyacceptable carrier to comprise a physiologically acceptable compositionfor administration. Such compositions comprise a therapeuticallyeffective amount of the FGF-22 polypeptide and a physiologicallyacceptablely acceptable carrier or excipient. Such a carrier includesbut is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration. Preferably the cells are located in tendons,ligaments, and synovial membranes. More specifically the cells would befibroblasts present in loose, dense, collagenous and elastic connectivetissues of the tendons and/or ligaments and/or synoviocytes withinsynovial membranes and contacted using the methods of Chan, et al. (ActaOrthop Scand 71: 513-518, 2000) and Kuroda, et al. (Knee Surg SportsTraumatol Arthrosc 8:120-126, 2000), which disclosures are herebyincorporated by reference in their entirety. More preferably thefibroblasts would be induced to actively synthesize dense connectivetissue and/or collagen. Preferably, the connective tissue is contactedby administering a FGF-22 polypeptide to an individual. Preferably, theFGF-22 polypeptide is administered parenterally, more preferablyintraperitoneal.

A further specified embodiment of the present invention is a method ofpromoting cartilage (hyaline cartilage, fibrocartilage, elasticcartilage) wound repair or tissue healing, in vitro and in vivo, such asresultant from aging, post-operative damage, disease, or other insult,by stimulating cartilage tissue cell proliferation or increasingcartilage tissue cell viability by contacting said cells, in vitro or invivo, with a proliferation stimulating or viability increasing effectiveamount of a FGF-22 polypeptide. The polypeptide of the present inventionmay be employed in combination with a suitable physiologicallyacceptable carrier to comprise a physiologically acceptable compositionfor administration. Such compositions comprise a therapeuticallyeffective amount of the FGF-22 polypeptide and a physiologicallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration. Preferably the cells are located within joints and/orarticular surfaces involved in interstitial/endogenous growth and/orappositional/exogenous growth, ends of long bones (articular cartilage),ends of ribs (costal cartilage), intervertebral disks, symphysis ofpubis, menisci of knee, nasal septum, larynx, pharynx, trachea, bronchi,epiglottis, sternum, Eustachian tubes, and of the external (pinna),middle, and inner ear. More specifically the cells would be groundsubstance (collagenous or elastic fibers, glycosaminoglycans,chondroitin sulfate matrix) remodeling cells(chondrocytes,chondroblasts, chondroclasts) present in cartilagenousconnective tissues and contacted using the methods of Toolan, et al. (JBiomed Mater Res 31: 273-280, 1996), Shida, et al. (J Orthop Res 14:265-272, 1996), and/or Chan, et al. (Clin Orthop 342: 239-247, 1997),which disclosures are hereby incorporated by reference in theirentirety. More preferably the cartilage cells (chondrocytes,chondroblasts) would be induced to actively synthesize ground substance.Preferably, the connective tissue is contacted by administering a FGF-22polypeptide to an individual. Preferably, the FGF-22 polypeptide isadministered parenterally, preferably intraperitoneal, or by anyconvenient manner, or by syringe or catheter at the location of targetedcartilage connective tissue biosynthesis (Chan et al., Clin Orthop 342:239-247, 1997).

A further specified embodiment of the present invention is a method ofpromoting osseous (compact bone, spongy bone) wound repair or tissuehealing, in vitro and in vivo, such as resultant from aging,post-operative damage, disease, or other insult, by stimulating osseousconnective tissue cell (osteoblast progenitor stromal stem cell,osteocyte, osteoblast, osteoclast) proliferation or increasing osseusconnective tissue cell viability by contacting said cells, in vitro orin vivo, with a proliferation stimulating or viability increasingeffective amount of a FGF-22 polypeptide. The polypeptide of the presentinvention may be employed in combination with a suitable physiologicallyacceptable carrier to comprise a physiologically acceptable compositionfor administration. Such compositions comprise a therapeuticallyeffective amount of the FGF-22 polypeptide and a physiologicallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration. Preferably the cells (osteoblast progenitor stromal stemcell, osteocytes, osteoblast) would be induced to actively synthesizeintestitial matrix substance containing mineral salts such as calciumphosphate and calcium carbonate as well as collagenous fibers. Theosseous tissue cells would be contacted using the methods of Mathijssen,et al. (J Craniofac Genet Dev Biol 20: 127-136, 2000), Reiff, et al. (JTrauma 50: 433-438, 2001) and/or Mackenzie, et al. (Plast Reconstr Surg107: 989-996, 2001). In response to FGF-22 treatment, radiomorphometric(percentage of radiopacity of defect) and histomorphometric (squaremillimeters of new bone formation) methods would be used to derivequantitative outcome data. bone formation). Preferably, the osseusconnective tissue is contacted by administering a FGF-22 polypeptide toan individual. Preferably, the FGF-22 polypeptide is administeredparenterally, preferably intraperitoneal, or by any convenient manner,or by syringe or catheter at the location of targeted osteosynthesis(Radomsky, et al., Clin Orthop 355 Suppl: S283-S293, 1998), or bydirected intraosseous injection using the methods of (Nakamura, et al.,J Orthop Res 15: 307-313, 1996; Nakamura et al., Int Orthop 22: 49-54,1998).

FGF-22 is expressed in the ventrolateral thalaric nucleus of the CNS, aregion associated with paralysis agitans, or Parkinson's Disease.Surgical intervention using thalamatomy for Parkinson's disease involvesintroduction of lesions in the ventrolateral thalamus to relieve tremorand improve rigidity. Therefore, a further embodiment of this inventionis a method of attenuating Parkinson's Disease associated tremors, orunrelated benign essential tremors, by contacting ventrolateral thalamictissue comprising the steps of contacting said cells with an effectiveamount of a FGF-22 polypeptide. Another aspect of the present inventionrelates to a method for enhancing and/or stimulating and/or maintainingand/or regenerating the formation and/or survival of neurons in vitro orin the central nervous system of a mammal which comprises contactingneurons or neural progenitor cells, e.g., in vitro or by administeringto said mammal, an effective amount of FGF-22 for a time and underconditions sufficient to effect an increase in and/or to maintain thenumber of neurons in the central nervous system. Prefereably the cellsand/or tissue is located within the thalamic region of the CNS. Morepreferably the cells and/or tissue are of the thalamic ventral nuclei.The polypeptide of the present invention may be employed in combinationwith a suitable physiologically acceptable carrier to comprise aphysiologically acceptable composition for administration. Suchcompositions comprise a therapeutically effective amount of the FGF-22polypeptide and a physiologically acceptablely acceptable carrier orexcipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.Preferably, the CNS tissue is contacted by administering a FGF-22polypeptide to an individual. Preferably, the FGF-22 polypeptide isadministered parenterally, with the route of administrationintraperitoneal, intramuscular, or by intravenous injection, or usinggene therapy, although additional routes are possible such as infusion,drip, intracerebral injection (Mufson, et al., Prog Neurobiol 57:451-484, 1999) and/or implants (Shults, et al., Brain Res 883: 192-204,2000; Tornqvist, et al., Exp Neurol 164: 130-138, 2000) and as describedin U.S. Pat. No. 6,179,826, which disclosures are hereby incorporated byreference in their entireties. FGF-22 may also be administered directlyto the brain. In an additional embodiment of this invention, FGF-22 mayalso be employed to stimulate neuronal growth and to treat and preventneuronal damage associated with stroke or which occurs in certainneuronal disorders or neurodegenerative conditions such as Alzheimer'sand AIDS-related complex.

The Adeno Associated Virus (AAV) utilizes the human FGFR-1 as aco-receptor for infection in mammalian cells (Qing, et al, Nat Med 5:71-77, 1999, which disclosures are hereby incorporated by reference intheir entirety) as well as the ubiquitously expressed heparan sulfateproteoglycans on cell surfaces. Similarly, adenoviral vectors areeffectively targeted for the treatment of systemic and local diseaseusing the ability of FGF family polypeptides to bind their cognateFGFR's with high affinity (Sosnowski, et al, Curr Opin Mol Ther 1:573-579, 1999, which disclosures are hereby incorporated by reference intheir entirety). As a further embodiment of this invention is a methodof retargeting a FGF-22 polypeptide or chimeric polypeptide encoded aspart of an adenoviral or AAV delivery system to cells expressing cognateFGFR complexes using the methods of Hoganson, et al ., (Mol Ther 3:105-112, 2001) and Qing, et al . (Nat Med 5: 71-77, 1999), whichdisclosures are hereby incorporated by reference in their entirety.Preferably the FGF-22 polypeptide is expressed, in part or in whole,with the viral delivery system as a bifunctional conjugate consisting ofa blocking anti-adenoviral knob Fab fragment linked to FGF-22 using themethods of Goldman, et al (Cancer Res 57:1447-51, 1997) and Doukas, etal. (FASEB J 13:1459-66, 1999). Preferably the FGFR complex is theFGFR-1 polypeptide or FGFR-1 polypeptide ligand binding moiety.

Protein of SEQ ED NO:18 (Internal Designation Clone229633_(—)253-2-5-2-A11-F)

The cDNA of Clone 229633_(—)253-2-5-2-A11-F (SEQ ID NO:17) encodes theSTAM-SAPper (STAMSAP) protein comprising the amino acid sequence:

MDRALQVLQSIDPTDSKPDSQDLLDLEDICQQMGPMIDEKLEEIDRKHSELSELNVKVLEALELYNKLVNEAPVYSVYSKLHPPAHYPPASSGVPMQTYPVQSHGGNYMGQSIHQVTVAQSYSLGPDQIGPLRSLPPNVNSSVTAQPAQTSYLSTGQDTVSNPTYMNQNSNLQSATGTTAYTQQMGMSVDMSSYQNTTSNLPQLAGFPVTVPAHPVAQQHTNYHQQPLL (SEQ ID NO:18).Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:18 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 229633_(—)253-2-5-2-A11-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:17 described throughout the present application alsopertain to the nucleic acids included in Clone229633_(—)253-2-5-2-A11-F. A preferred embodiment of the invention isdirected toward the compositions comprising SEQ ID NO:17, SEQ ID NO:18,and Clone 229633_(—)253-2-5-2-A11-F. Another preferred embodiment of theinvention is directed toward compositions comprising polynucleotidefragments of at least eighteen contiguous nucleotides selected from:gagcaagacgtggtgatgccaattggtggaaaggagaaaatcac, preferably thosepolynucleotides that encode for polypeptides having a biologicalactivity described herein. Further preferred polynucleotides of thepresent invention include nucleic acids comprising:gaagcggmgsggtctagggagccgccggccgcgggtcacccggcgggtagcagttgctgagtgtcagtagacagcagcgactagggctcgggcgccggcgagatgcctttgttcaccgccaaccccttcgagcaagacgtggtgatgccaattggtggaaaggagaaaatcacpreferably those that encode for polypeptides having a biologicalactivity described herein. Further preferred polynucleotides of thepresent invention include nucleic acids of SEQ ID NO:17 comprisinggaagcggmgsggtctagggagccgcggccgcgggtcacccggcgggtagcagttgctgagtgtcagctagacagcagcgactagggctcgggcgccggcgagatgcctttgttcaccgccaaccccttcgagcaagacgtggtgatgccaattggtggaaaggagaaaatcacagaggaataggacttttcccatccaattttgtaacaactaatttaaacatagagactgaggcagcggctgtggacaaattgaatgtaattgatgatgatgtggaggaaattaagaaatcagagcctgagcctgtttatatagatgaggataagatggatagagccctgcaggtacttcagagtatagatccaacagattcaaaaccagactcccaagaccttttggatttagaagatatctgccaaca preferably thosethat encode for polypeptides of having a biological activity describedherein. Polypeptides of the invention having a biological activity of x%, where x is any integer between 1 and 100 of those described herein andpolynucleotides encoding the same are also included in the invention.Polypeptides of the invention with biological activity are defined aspolypeptides that can be phosphorylated by a tyrosine kinase such as aJanus kinase (Jak).

STAMSAP protein results from a splice event within the SignalTransducing Adaptor Molecule (STAM)-2 transcript. This splice variantcontacts or recombines nucleotide 152 of STAM-2 with nucleotide 817. Theresulting STAMSAP splice variant encodes the carboxy-terminal 228 aminoacids of the 525-amino acid STAM-2 protein. STAM-2 contains threewell-characterized domains. The first is an SH3 domain spanning aminoacids 212-266 that is not shared with STAMSAP. This SH3 domain binds thedownstream effector of STAM-2, AMSH, which activates proto-oncogenictranscription factors comprising c-myc and AP-1, and results inresponses that include cell proliferation (Tanaka, N., et al. (1999) J.Biol. Chem. 274: 19129-35 which disclosure is hereby incorporated byreference in its entirety). An Immunoreceptor Tyrosine-based ActivationMotif (ITAM) spanning amino acids 359-387 of STAM-2 and acarboxy-terminal tyrosine-rich domain are shared with STAMSAP (Endo, K.,et al. (2000) FEBS Let. 477:55-61 and Pandey, A., et al. (2000) J. Biol.Chem. 275:38633-9 which disclosures are hereby incorporated by referencein their entireties).

STAMSAP is phosphorylated on tyrosine residues within the ITAM andcarboxy-terminal domains by Jak molecules comprising Jak2 and Jak3. Jak2and Jak3 phosphorylate STAMSAP in response to ligand binding of cellsurface receptors comprising IL-2R, IL-3R, IL-4R, IL-7R, PlateletDerived Growth Factor Receptor (PDGFR), Epidermal Growth Factor Receptor(EGFR), and Granulocyte Macrophage Colony Stimulating Factor Receptor(GM-CSFR). Jak activation and subsequent gene expression is associatedwith proliferation and cancers comprising breast and colon carcinomasand B cell lymphomas (Yamauchi, T., et al. (2000) J. Biol. Chem.275:33937-44; Kaulsay, K., (2000) Endocrinology 141:1571-84; U.S. Pat.No. 6,177,433 which disclosures are hereby incorporated by reference intheir entireties). Jak is often hyperactivated due to abnormally highexpression of upstream receptors or their ligands in cancer cells. Forexample, higher than normal levels of PD GF are indicative of advancedstages of breast cancer (Seymour, L., et al. (1993) Breast Cancer Res.Treat. 26:247-52 which disclosure is hereby incorporated by reference inits entirety). EGFR is overexpressed in a variety of tumors includingcervical cancer (Mathur, R., et al. (2000) Am. J. Reprod. Immunol.44:114-20 which disclosure is hereby incorporated by reference in itsentirety). Furthermore, Jak3 is activated in stimulated mast cells,causing degranulation and subsequent allergic reactions (U.S. Pat. No.6,177,433 which disclosure is hereby incorporated by reference in itsentirety).

STAMSAP does not have a downstream effector and therefore acts as adominant negative inhibitor of Jak signaling. In a preferred embodimentof the invention, the STAMSAP polypeptide is used to inhibit cellproliferation, cell survival, or viral replication downstream of Jaksignaling. This embodiment is accomplished by methods comprising thestep of delivering STAMSAP to cells responsive to activated Jak, forexample, MOLT4 cells expressing IL-2R (ATCC number CRL-1582). Methodsfor delivering STAMSAP to Jak-resposive cells include contacting saidcells with STAMSAP polynucleotides or polypeptides by methods common tothe art as discussed in the following paragraph. Further included inthis embodiment is a polynucleotide comprising polynucleotides encodinga STAMSAP polypeptide with biological activity operably linked to anexpression control element such as a promotor. Said polynucleotide isdelivered to Jak-responsive cells by methods common to the art such aselectroporation or transfection of naked polynucleotides. In addition,genes activated by Jak signaling may be monitored or assayed usingmethods common to the art, for example, reporter gene assays such asluciferase or beta-galactosidase. This embodiment is applied to, forexample, inhibiting Jak-dependent cell responses in vitro.

Another preferred embodiment of the invention is directed towardsmethods to use STAMSAP to inhibit Jak-induced cell proliferation. Inparticular, this embodiment is directed toward inhibiting proliferationof cells resulting from activation of any upstream effector of Jak, suchas a growth factor. Preferred upstream effector molecules include butare not limited to: PDGFR, EGFR, IL-2R, IL-3R, IL-4R, IL-7R, andGM-CSFR. STAMSAP is used in this method comprising the step ofintroducing a STAMSAP polypeptide or a polynucleotide comprisingpolynucleotides encoding said polypeptide operably linked to anexpression control element into cells activated by Jak or any upstreameffector of Jak (e.g., cervical cancer cells stimulated with EGF).Preferred control elements express an amount of STAMSAP effective toinhibit proliferation of cells to which the invention is delivered.Alternative preferred control elements comprise cell- or tissue-specificenhancer elements, for example, the lyn enhancer for B cells, or c-mycor AP-1 sites for proliferating cells. Said polypeptides orpolynucleotides are introduced into said cells using methods common tothe art, including but not limited to lipid vesicles or viraltransduction, as described in any one of the list: U.S. Pat. Nos.5,616,565, 6,110,490, and 6,204,060, or WO9704748 which disclosures arehereby incorporated by reference in their entireties. For example,polynucleotides are delivered to said cells by: i) compressing apolynucleotide expression unit, preferably an expression unit containingpolynucleotides encoding biologically active STAMSAP polypeptide, into alipid vesicle derived from any of the following list: viral envelopes,liposomes, micelles, and modified versions of these, as described inU.S. Pat. No. 6,110,490 or P.C.T. 904748, which disclosures are herebyincorporated by reference in their entireties; ii) optionally targetingthe lipid vesicle to specific cells, for example, by embedding a memberof a receptor-receptor ligand pair into the lipid envelope (e.g., CD40ligand for targeting to B cells); iii) contacting the targeted vesiclewith specific cells by methods common to the art such as injection orinhalant (U.S. Pat. No. 6,110,490, P.C.T 9704748, and U.S. Pat. No.6,034,062 which disclosures are hereby incorporated by reference intheir entireties). An example of delivering polypeptides to said cellscomprises the steps: i) packaging a biologically active STAMSAPpolypeptide into a lipid vesicle; ii) targeting the lipid vesicle tospecific cells, for example, by including a member of areceptor-receptor ligand pair in the lipid envelope; iii) embedding afusogenic component such as a peptide in the lipid envelope to promotedelivery of encapsulated polypeptides to target cells; and iv)contacting the targeted vesicle with specific cells by injection orinhalant (P.C.T. 9704748 and U.S. Pat. No. 6,034,062 which disclosuresare hereby incorporated by reference in their entireties).

In another preferred embodiment, STAMSAP is used to inhibit Jak3 incells that induce an inflammatory response, such as mast cells,eosinophils, T cells, and B cells. This embodiment includes a method todeliver a biologically active STAMSAP polypeptide or a polynucleotidecomprising polynucleotides encoding said polypeptide operably linked toan expression control element to individuals displaying the effects ofan inflammatory response (e.g., allergic rhinitis (hay fever), allergicurticaria (hives), angioedema, allergic asthma, or anaphylaxis).Preferred methods of delivery include but are not limited to a methodcomprising the steps: i) packaging of said polynucleotide into a lipidvesicle as described in U.S. Pat. Nos. 6,110,490, 5,616,565, and P.C.T.9704748 which disclosures are hereby incorporated by reference in theirentireties, and ii) delivering the vesicle to cells that induce anallergic response, such as mast cells, so that STAMSAP polypeptide iscontacted with the relevant intracellular site. Preferred controlelements direct expression of an amount of STAMSAP effective to inhibitan inflammatory response. Further preferred control elements for use inthis embodiment include promoters of cell-specific genes such as CD48 inmast cells. The lipid vesicle is derived from any of the following list:viral envelopes, liposomes, micelles, and modified versions of these.Targeting of vesicles to specific cell types, as referred to in step(ii), is effected by embedding a targeting moiety such as a member of areceptor-receptor ligand pair into the lipid envelope of the vesicle.Useful targeting moieties specifically bind cell surface ligands, suchas CD48 or the SCF receptor on mast cells. Thus, anti-CD48 antibodies orSCF ligand are examples of useful mast cell-targeting moieties. Inaddition, the antibodies B43 and TXU are useful for B and T cells,respectively. Vectors and targeting are further described in U.S. Pat.Nos. 6,177,433, 6,110,490, and P.C.T. 9704748, which disclosures arehereby incorporated by reference in their entireties. The invention isdelivered to the appropriate site by methods common to the art such asinjection or inhalant as described in U.S. Pat. Nos. 6,177,433 and6,034,062, which disclosures are hereby incorporated by reference intheir entireties.

Protein of SEQ ID NO:22 (Internal Designation Clone589198_(—)184-11-1-0-E4-F)

The cDNA of Clone 589198_(—)184-11-1-0-E4-F (SEQ ID NO:21) encodes theCorneal Osteo-Vascular Inducing (COVI) protein comprising the amino acidsequence:

MKTLQSTLLLLLLVPLIKPAPPTQQDSRIIYDYGTDNFEESIFSQDYEDKYLDGKNIKEKETVIIPNEKSLQLQKDEAITPLPPKKENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPKESAYLYARFNKIKKLTAKDFADIPNLRRLDFTGNLIEDIEDGTFSKLSLLEELSLAENXLLKLPVLPPKLTLFNAKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLPESLRVIHLQFNNIASITDDTFCKANDTSYIRDRIEEIXLEGNPIVLGKHPNSFICLKRLPIGSYF (SEQ ID NO:22).Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:22 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 589198_(—)184-11-1-0-E4-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:21 described throughout the present application alsopertain to the nucleic acids included in Clone589198_(—)184-11-1-0-E4-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:21, SEQ ID NO:22, andClone 589198_(—)184-11-1-0-E4-F. Further included in the invention arepolypeptide fragments at least seven amino acids in length of SEQ IDNO:22 and those having a biological activity of those described hereinand polynucleotides encoding the same. Biological activities include butare not limited to increasing bone density when contacted withosteogenic cells and remodeling of vascular tissue.

The COVI polypeptide is a unique splice variant of the mimecan (alsocalled osteoglycin and osteoinductive factor) gene (Kukita, A., et al.(1990) Proc. Natl. Acad. Sci. 87:3023-6, Funderburgh, J., et al. (1997)J. Biol. Chem. 272:28089-95, and Tasheva, E., et al. (1999) J. Biol.Chem. 274:18693-701 which disclosures are hereby incorporated byreference in their entireties). The 1997 base pair COVI transcriptbegins in exon 3 of full-length mimecan and encodes a 298 amino acidprotein.

The COVI polypeptide is a secreted protein associated with theextracellular matrix (ECM) that promotes growth and remodeling of bone.In a preferred embodiment of the invention, COVI polypeptide is used ina method to promote bone growth by contacting a bone growth-stimulatingeffective amount of COVI polypeptide with cells. Preferred cells arethose that normally produce bone tissue, including but not limited toosteoblasts, osteocytes, and their precursors. This method is useful tofacilitate bone growth in cases including but not limited to bone loss,atrophy, or malformation due to injury, congenital or chronicconditions, surgery, or disease. Examples include but are not limited toosteopenia, osteoporosis, rickets, malignant melanoma-induced bonedegradation, and bone fissures or fractures due to injury, electivesurgery (e.g., plastic surgery), reconstructive surgery, and dentalprocedures or surgeries. COVI polypeptides are delivered in aphysiologically acceptable solution, for example, pH-buffered saline,viscous solutions such as those including glycerol or dextrose, or insolutions that include other components to support bone growth.Preferred bone growth components comprise bone fragments, ground bone,and matrix materials including calcium sulfate, hydroxyapatite,ultrahigh molecular weight polyethylene (UHMWPE), and proteins such ascollagen. COVI polypeptides in physiologically acceptable solution aredelivered locally or systemically (as the case dictates) by methodsincluding but not limited to injection, catheter delivery, or directimplantation (U.S. Pat. No. 6,034,062 which disclosures are herebyincorporated by reference in their entirety).

A further embodiment of the invention is a method of contacting a bonegrowth-stimulating amount of COVI polypeptide with cells to facilitateintegration of bone, for example, for purposes of bone transplantationin cases of dental implants, orthopedic prosthesis, or other surgicalprocedures. Preferred cells are those present in bone tissue, includingbut not limited to osteoblasts, osteocytes, and their precursors. COVIpolypeptides are delivered in a physiologically acceptable solution, forexample, pH-buffered saline, viscous solutions such as those includingglycerol or dextrose, or in solutions that include other components tosupport bone growth. Preferred bone growth components comprise bonefragments, ground bone, and matrix materials including calcium sulfate,hydroxyapatite, UHMWPE, and proteins such as collagen. COVI polypeptidesin physiologically acceptable solution are delivered to the site ofdesired bone integration by methods comprising injection or directaddition to the integrated tissue (U.S. Pat. No. 6,034,062, whichdisclosure is hereby incorporated by reference in its entirety).

A further embodiment of this invention is a method of contacting agrowth-stimulating amount of COVI polypeptide with cells to facilitatebone growth for example, for purposes of transplantation. Preferredcells include bone cells. Further preferred cells include but are notlimited to human osteoblast cells, for example the cell lines MG63 orC2C12 or osteoblasts purified directly from bone, or their progenitors,such as those purified from bone marrow stroma or mesenchymal stemcells. Preferred culture conditions are common to the art and caninclude but are not limited to other factors to promote bone formation,for example bone or composite matrices to direct shaping, ascorbic acid,beta-glycerophosphate, dexamethasone, calcium salts, and collagen [Dean,D., et al. (2001) J. Orthop. Res. 19:179-86 and Buttery, L., et al.(2001) Tissue Eng. 7:89-99, which disclosures are hereby incorporated byreference in their entireties]. A preferred method comprises the steps:contacting COVI polypeptide directly with cells in culture; harvestingmineralized bone formation; and surgically implanting newly formed boneinto desired location (U.S. Pat. Nos. 4,950,296, 5,385,566, and6,200,324, which disclosures are hereby incorporated by reference intheir entireties). Another preferred method comprises the steps:delivering polynucleotides to cells in culture; delivering cells tosites of desired bone growth (for example, to the site of a fracture orto an osteopenic bone). Preferred polynucleotides comprisepolynucleotides encoding COVI polypeptide operably linked to anexpression control unit (e.g., a promoter) that will deliver a bonegrowth-stimulating amount of COVI expression (for example, high,constitutive expression from the CMV promoter or regulated expressionfrom a tetracycline-repressible promoter, both of which are readilycommercially available). Said polynucleotides are delivered to cells invitro or in situ by methods common to the art such as electroporation,calcium phosphate transfection, or adenoviral transduction [Maniatis,T., et al. Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory (1982) and Cheng, S., et al. (2001) Calcif. Tissue Int.68:87-94, which disclosures are hereby incorporated by reference intheir entireties]. Cells are introduced to a site of desired bone growthin vitro, in situ, or in vivo by methods comprising injection,introduction through a catheter, or surgical implantation of acell-containing stent, for example, on an osteopenic bone (U.S. Pat.Nos. 6,034,062 and 6,206,914, which disclosures are hereby incorporatedby reference in their entireties).

COVI is associated with vascular smooth muscle cells (VSMC) in the ECM.The COVI splice variant has enhanced ability to promote vascular matrixremodeling, i.e., formation of new vessels (e.g., during development ortissue expansion), and healing of damaged vessels such as thoseresulting from injury, incision, burns, disease, cardiac infarction,ulcers, diabetic ulcers, and chronic conditions such as atherosclerosis.A preferred embodiment of the invention is a method to promote vascularremodeling by contacting a vascular remodeling-stimulating amount ofCOVI polypeptide with cells. Preferred cells include but are not limitedto VSMC, vascular epithelial cells, and fibroblasts. Further preferredcells include but are not limited to human VSMC, vascular epithelialcells, and fibroblasts in intact tissue (i.e., in a milieu of ECMproteins such as collagen). COVI polypeptides are delivered to cells inphysiologically acceptable solution, for example, pH-buffered saline orviscous solutions such as those including glycerol or dextrose. Saidsolution may be applied topically to surface wound tissue in thetreatment of ulcers, lesions, injuries, diabetic ulcers, bums, trauma,stasis ulcers, periodontal conditions, lacerations, and otherconditions. In addition, intraperitoneal wound tissue such as thatresulting from invasive surgery may be treated with a physiologicallyacceptable solution comprising COVI polypeptides to accelerate vascularremodeling. For example, the surgical plane may be coated with saidsolution prior to closing the surgical site to facilitate internalcapillary perfusion and healing. In addition, the rate of localizedhealing may be increased by the subdermal administration of saidsolution by methods common to the art such as injection (U.S. Pat. No.6,096,709, which disclosure is hereby incorporated by reference in itsentirety).

Timely vascular remodeling is an urgent factor in the case of cardiacinfarction to prevent enlargement of the organ. A further preferredembodiment of the invention is a method of contacting a vascularremodeling-stimulating amount of COVI polypeptide with cells. The methodcomprises the step of contacting COVI polypeptides with cells byimplantation of a COVI polypeptide-releasing stent, for examplesurgically or via catheter (U.S. Pat. Nos. 5,500,013 and 5,449,382,which disclosures are hereby incorporated by reference in theirentireties). Preferred cells include but are not limited to those foundin cardiac tissue damaged as a result of infarction or within vesselsfor treating various problems such as atherosclerosis, stenonses,strictures, or aneurysms to reinforce collapsing, partially occluded, orweakened sections.

A further preferred embodiment of the invention is a method to promotevascular remodeling by delivering polynucleotides encoding COVIpolypeptides to cells. This method is directed toward purposes such astransplantation of cells expressing COVI polypeptides. Preferred cellsinclude but are not limited to VSMC, vascular epithelial cells, andfibroblasts. Further preferred cells include but are not limited tohuman VSMC, vascular epithelial cells, and fibroblasts, preferably inintact tissue (i.e., in a milieu of ECM proteins such as collagen).Preferred polynucleotides comprise polynucleotides encoding COVIpolypeptides operably linked to an expression control unit (e.g., apromoter) that will deliver a vascular remodeling-stimulating amount ofCOVI expression (for example, high, constitutive expression from the CMVpromoter or regulated expression from a tetracycline-repressiblepromoter, both of which are readily commercially available). Saidpolynucleotides are delivered to cells in vitro or in situ by methodscommon to the art such as electroporation, calcium phosphatetransfection, or adenoviral transduction [Maniatis, T., et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(1982) and Cheng, S., et al. (2001) Calcif. Tissue Int. 68:87-94, whichdisclosures are hereby incorporated by reference in their entireties].Further included in the method is a step of delivering said cells to adesired site of vascular remodeling (including but not limited towounds, incisions, injuries, ulcers, and diseased or otherwisehypovascular lesions) by methods common to the art such as injection orcatheter delivery of cell suspensions or surgical implantation of intacttissue endoscopically or invasively (U.S. Pat. Nos. 5,669,925 and5,683,345, which disclosures are hereby incorporated by reference intheir entireties). COVI polypeptide is also present as a highly modifiedkeratan sulfate proteoglycan (KSPG) in the cornea. KSPG's are associatedwith ECM proteins in the cornea and function to maintain corneal shapeand opacity. In a further embodiment of the invention, acornea-maintaining effective amount of COVI polypeptide is used in amethod for maintaining a desired shape (e.g., following laser surgery ornon-invasive orthokeratological procedures) or opacity of cornealtissues (e.g., at the onset of cataract formation). This methodcomprises the step of contacting COVI polypeptides with the ECM of thecornea in a physiologically acceptable solution. A preferredphysiologically acceptably solution includes pH-buffered saline.Preferred method of contact is by an eye-drop mechanism (P.C.T.00119386, which disclosure is hereby incorporated by reference in itsentirety).

Protein of SEQ ID NO:4 (Internal Designation Clone1000848582_(—)181-40-4-0-A11-F)

The cDNA of clone 1000848582_(—)181-404-0-A11-F (SEQ ID NO:3) encodesthe protein of SEQ ID NO:4 comprising the amino acid sequence

MELALRRSPVPRWLLLLPLLLGLNAGAVIDWPTEEGKEVWDYVTVRKDAYMFWWLYYATNSCKNFSELPLVMWLQGGPGGSSTGFGNFEEIGPLDSDLKPRKTTWLQAASLLFVDNPVGTGFSYVNGSGAYAKDLAMVASDMMVLLKTFFSCHKEFQTVPFYIFSESYGGKMAAGIGLELYKAIQRGTIKCNFAGVALGDSWISPVDSVLSWGPYLYSMSLLEDKGLAEVSKVAEQVLNAVNKGLYREATELWGKAEMIIEQVKRGNTQRLACLAFSGGYRAHGWCCQTWSLH. Accordinglyit will be appreciated that all characteristics and uses of polypeptidesof SEQ ID NO:4 described throughout the present application also pertainto the polypeptides encoded by the nucleic acids included in Clone1000848582_(—)181-40-4-0-A11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:3described throughout the present application also pertain to the nucleicacids included in Clone 1000848582_(—)181-40-4-0-A11-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:3, SEQ ID NO:4, and Clone 1000848582_(—)181-40-4-0-A11-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:4 encodes a novel serine carboxypeptidasedesignated here serine carboxypeptidase hx (SCPhx). SCPhx has a uniqueC-terminal sequence of 31 amino acids comprisingKRGNTQRLACLAFSGGYRAHGWCLQTWSLH. This unique sequence within SCPhxcontributes the histidine of the catalytic triad. SCPhx cleaves thepeptide bond between the penultimate and C-terminal amino acid residuesof its protein or peptide substrate and, in so doing, can eitheractivate or inactivate the biological function of the substrate.

A preferred embodiment of the invention is directed to compositionscomprising the amino acid sequence of SEQ ID NO:4 (SCPhx) or fragmentsthereof. Further preferred is a method to use the serinecarboxypeptidase activity of compositions comprising SCPhx polypeptidefor biosynthetic procedures. Further preferred is an application of saidmethod wherein a recombinant polypeptide engineered with a protectivebut inactivating C-terminal amino acid is activated through removal ofthis amino acid by SCPhx.

Further preferred is a method to use the serine carboxypeptidaseactivity of compositions comprising SCPhx polypeptide for analyticalprocedures. Further preferred is an application of said method whereinthe requirement for the C-terminal amino acid for the function of agiven protein is determined through removal of the amino acid by SCPhx.

The serine carboxypeptidase activity of SCPhx confers on SCPhxantifibrinolytic activity. In a further embodiment, compositions of theinvention comprised of SCPhx are used in methods wherein theantifibrinolytic activity of SCPhx is used to promote wound healing. Infurther preferred embodiment, the composition is used in methods ofstabilizing blood clots at sites where there is a breach in thevasculature by contacting a wound or injured tissue with aregenerative-effective amount of compositions of the invention.

In a further embodiment of the invention, SCPhx is used in a method forantibody-directed enzyme prodrug therapy (ADEPT). In said method, invivo localization of SCPhx serine carboxypeptidase activity is effectedthrough conjugation of SCPhx to specific antibody. Injection ofSCPhx-antibody conjugate in conjunction with prodrug(drug-alpha-peptide) (Shi, P. T., et al., Yao Xue Bao 32:106-9 (1997)which disclosure is hereby incorporated by reference in its entirety)results in localized activation of the drug.

In said method for ADEPT, a preferred embodiment of the invention isdirected to compositions comprising SCPhx conjugated to tumor-reactiveantibody [Napier, M. P., et al., Clin. Cancer Res. 6:765-72 (2000) whichdisclosure is hereby incorporated by reference in its entirety]. Infurther preferred embodiment, SCPhx is conjugated to antibody reactivewith carcinoembryonic antigen (CEA) and is used in conjunction withmethotrexate prodrug for the treatment of colorectal carcinoma.

In a further preferred embodiment, the present invention provides for anantibody that binds SCPhx with or without neutralization of SCPhx serinecarboxypeptidase activity. The antibody may be monoclonal or polyclonal.Preferred compositions comprise the SCPhx antibody.

SCPhx serine carboxypeptidase activity expressed by breast cancer cellscan activate autocrine neuropeptide growth factors concomitantlyexpressed by the tumor cells. In further embodiment of the invention,neutralizing anti-SCPhx antibody is used by intravenous injection tosuppress tumor growth by blocking the activation of autocrine growthfactors by SCPhx constitutively expressed by the tumor. In furtherpreferred embodiment, said method is used for the treatment of breastcancer. In further preferred embodiment, said method is used for thetreatment of cancer of the salivary gland.

SCPhx serine carboxypeptidase activity can process beta-amyloidprecursor protein and generate beta-amyloid. In further embodiment ofthe invention, neutralizing anti-SCPhx antibody is used by injection inAlzheimer's disease to block processing of beta-amyloid precursorprotein and generation of beta-amyloid.

Daily administration of a very low dose of the polypeptide gAcrp30 tomice consuming a high-fat/sucrose diet causes profound and sustainableweight reduction without affecting food intake (Fruebis, J., et al.,Proc. Natl. Acad. Sci. USA 98:2005-10 (2001) which disclosure is herebyincorporated by reference in its entirety). Said activity of gAcrp30 isabrogated by SCPhx serine carboxypeptidase activity. In a preferredembodiment of the invention, compositions comprising said neutralizingSCPhx antibody are used in methods to block in vivo inactivation ofpolypeptide function by SCPhx serine carboxypeptidase activity. Infurther preferred embodiment, compositions comprising said neutralizingSCPhx antibody are used in methods to treat obesity in humans byintravenous injection concomitant with human gAcrp30. In furtherpreferred embodiment, compositions comprising said neutralizing SCPhxantibody are used in methods to treat obesity in other mammals byintravenous injection concomitant with mammal or human gAcrp30.

The invention further relates to a method of screening for testcompounds that bind and/or inhibit SCPhx serine carboxypeptidaseactivity above comprising the steps of contacting an SCPhx polypeptidewith said test compound and detecting or measuring whether said testcompound binds said SCPhx polypeptide. Alternatively, the methodcomprises the steps of contacting an SCPhx polypeptide with substrate ofsaid SCPhx polypeptide in the presence of test compound and detecting ormeasuring the release of the C-terminal amino acid from said SCPhxsubstrate, wherein a difference in the amount of said release relativeto the amount of release in the absence of the test compound modulates,preferably inhibits, the serine carboxypeptidase activity of SCPhx.

Protein of SEQ ID NO:8 (Internal Designation Clone1000770704_(—)208-27-3-0-G6-F)

The cDNA of clone 1000770704_(—)208-27-3-0-G6-F (SEQ ID NO:7) encodesthe protein of SEQ ID NO:8 comprising the amino acid sequence

MRLPAQLLGLLMLWVSGSSGDIVMTQSPLFLPVTPGEPASISCRSSQSLLHVQGSNYLDWYHQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGPGTRVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. Accordinglyit will be appreciated that all characteristics and uses of polypeptidesof SEQ ID NO:8 described throughout the present application also pertainto the polypeptides encoded by the nucleic acids included in Clone1000770704_(—)208-27-3-0-G6-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:7described throughout the present application also pertain to the nucleicacids included in Clone 1000770704_(—)208-27-3-0-G6-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:7, SEQ ID NO:8, and Clone 1000770704_(—)208-27-3-0-G6-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:8 encodes the polypeptide CaIX, which bindsparathyroid hormone related protein (PTHrP), a hormone involved in bonemetabolism.

PTHrP was initially discovered as a tumor-derived systemic factor thatcauses humoral hypercalcemia of malignancy (HHM). PTHrP is now known toplay a major role in HHM. It has been identified as the major causativeagent in tumors that were previously thought to cause hypercalcemiathrough skeletal metastatic involvement. Hypercalcemia is the mostcommon life-threatening metabolic disorder associated with neoplasticdiseases, occurring in an estimated 10% to 20% of all persons withcancer. That PTHrP is not just a bystander but is the cause of thehypercalcemia is indicated by the observation that in animals withhypercalcemia caused by xenografts of human tumors, the infusion ofneutralizing antibodies to PTHrP reverses the hypercalcemia.

CalX binds to and neutralizes the activity of PTHrp, including theinduction of HHM. A preferred embodiment of the invention is directed tocomprising the amino acid sequence of SEQ ID NO:8 (CalX). Furtherincluded in the invention are fragments of full-length CalX having abiological activity described herein as well as the polynucleotidesencoding these fragments.

In a preferred embodiment, compositions of the invention are used inmethods to neutralize PTHrP, wherein compositions comprising CalX arecontacted with and thereby block PTHrP activity. A further embodiment isdirected toward a method to use compositions of CalX to suppress HHM. Infurther preferred embodiment, CalX is used to suppress HHM associatedwith breast cancer, pancreatic adenocarcinoma, prostate cancer, squamouscell carcinoma of lung, renal cell carcinoma, ovarian carcinoma, and Tcell leukemia/lymphoma.

It is believed that PTHrP plays a role in the pathophysiology associatedwith osteoarthritis. In further preferred embodiment, CalX is used in amethod to suppress bone resorption within an affected joint, preferablyin the synovium of a joint capsule. Said methods comprise contactingCalX compositions with the synovial fluid of the joint capsule.Preferred delivery of CalX includes injection or transdermal contact atthe site of the joint.

It is believed that PTHrP plays a role in the pathophysiology associatedwith rheumatoid arthritis. In further preferred embodiment, CalX is usedin a method to decrease inflammation within an affected joint,preferably in the synovium of a joint capsule. In further preferredembodiment, CalX is used in a method to decrease bone resorption withinan affected joint, preferably in the synovium of a joint capsule. Saidmethods comprise contacting CalX compositions with the synovial fluid ofthe joint capsule. Preferred delivery of CalX includes injection ortransdermal contact at the site of the joint.

Protein of SEQ ID NO:6 (Internal Designation Clone1000839315_(—)220-26-1-0-F3-F)

The cDNA of clone 1000839315_(—)220-26-1-0-F3-F (SEQ ID NO:5) encodesthe protein of SEQ ID NO:6 comprising the amino acid sequence:

MKFFVFALVLALMISMISADSHEKRHHGYRRKFHEKHHSYHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT. Accordingly it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:6 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone1000839315_(—)220-26-1-0-F3-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:5described throughout the present application also pertain to the nucleicacids included in Clone 1000839315_(—)220-26-1-0-F3-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:5, SEQ ID NO:6, and Clone 1000839315_(—)220-26-1-0-F3-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:6 encodes Chimerin, a chimeric polypeptideencoded by an exon derived from the histatin 1 gene spliced downstreamonto an exon derived from the linked statherin gene. Specifically, anexon encoding the N-terminal amino acids of both histatin 1 and Chimerin(MKFFVFALVLALMISMISADSHEKRHHGYRRKFHEKHHS) is spliced onto astatherin-derived exon that encodes the novel C-terminal amino acids ofChimerin (YHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT) but incontradistinction entirely 3′-untranslated nucleotide sequence instatherin mRNA.

Chimerin is a low molecular weight, histidine-rich salivary polypeptide.Chimerin functions as part of the nonimmune host defence system in theoral cavity.

Chimerin possesses broad spectrum antifungal activity, including thatagainst the pathogenic yeast Candida alibcans, with minimal cytotoxicitytowards normal host cells, suggesting its high potential as a novelanti-fungal therapeutic agent. Chimerin also possesses anti-bacterialactivity, including that against Streptococcus mutans strains and theperiodontopatheogenic Porphyromonas gingivalis. A great benefit ofChimerin is that to date no resistant fungal strains have beendemonstrated and moreover, that Chimerin can be hydrolyzed in a naturalway in the digestive tract. Therefore, Chimerin might be applied forlong term use, intermitting the application of antibiotics.

A preferred embodiment of the invention is directed to compositionscomprising the amino acid sequence of SEQ NO:6 (Chimerin)

MKFFVFALVLALMISMISADSHEKRHHGYRRKFHEKHHSYHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT.

Further included in the invention are fragments of the full-lengthChimerin polypeptide having a biological activity described herein aswell as the polynucleotides encoding these fragments. Preferredfragments with biological activity include the amino acid sequencecomprising

DSHEKRHHGYRRKFHEKHHSYHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT or

DSHEKRHHGYRR or

KFHEKHHSYHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT.

Further preferred is a method to use formulations comprising Chimerin ina physiologically compatible solution as further described in U.S. Pat.No. 4,725,576 (“Fungicidal polypeptide compositions containingL-histidine and methods for use therefore”) and incorporated bereference in its entirety, including but not limited to theincorporation of Chimerin into a mouth wash.

Further preferred is a method to use compositions comprising Chimerin asagents with which to treat a fungal or bacterial infection as furtherdescribed in U.S. Pat. No. 5,912,230 (“Anti-fungal and anti-bacterialhistatin-based peptides”) and incorporated by reference in its entirety.The said method is comprised of contacting said fungi and bacteria withan effective amount of Chimerin polypeptide of the present invention.Said method for treating a fungal or bacterial infection of claim isapplicable when said fungal or bacterial infection is selected from thegroup consisting of: (a) an infection of the oral cavity; (b) aninfection of the vagina; (c) an infection of the urethra; (d) aninfection of the ear; (e) an infection of the skin; (f) a respiratoryinfection; (g) a mucosal infection; (h) an ophthalmic infection; and (i)systemic infection.

Further preferred is a method to use compositions comprising Chimerin asdescribed as agents with which to prevent recurring fungal or bacterialinfection in patients including, but not limited to, those from thegroup consisting of: AIDS patients; diabetics; and xerostomia patients,including patients with Sjogren's syndrome and those patients whosesalivary gland function has been compromised as a result of radiationtherapy.

Further preferred is method to use compositions comprising Chimerin fortreating a fungal or bacterial infection wherein the fungus or bacteriumis selected from the group consisting of: (a) Candida albicans; (b)Actinomyces actinomycetemcomitans; (c) Actinomyces viscosus; (d)Bacteroides forsythus; (e) Bacteriodes fragilis; (f) Bacteriodesgracilis; (g) Bacteriodes ureolyticus; (h) Campylobacter concisus; (i)Campylobacter rectus; (j) Campylobacter showae; (k) Campylobactersputorum; (l) Capnocytophaga gingivalis; (m) Capnocytophaga ochracea;(n) Capnocytophaga sputigena; (o) Clostridium histolyticum; (p)Eikenella corrodens; (q) Eubacterium nodatum; (r) Fusobacteriumnucleatum; (s) Fusobacterium periodonticum; (t) Peptostreptococcusmicros; (u) Porphyromonas endodontalis; (v) Porphyromonas gingivalis;(w) Prevotella intermedia; (x) Prevotella nigrescens; (y)Propionibacterium acnes; (z) Pseudomonas aeruginosa; (aa) Selenomonasnoxia; (bb) Staphylococcus aureus; (cc) Streptococcus constellatus; (dd)Streptococcus gordonii; (ee) Streptococcus intermedius; (ff)Streptococcus mutans; (gg) Streptococcus oralis; (hh) Streptococcuspneumonia; (ii) Streptococcus sanguis; (kk) Treponema denticola; (ll)Treponema pectinovorum; (mm) Treponema socranskii; (nn) Veillonellaparvula; and (oo) Wolinella succinogenes.

The compositions and methods for treatment of fungal and bacterialinfections discussed above are not limited to use in humans, but canhave veterinary applications as well.

In a further preferred embodiment, the present invention provides for anantibody that specifically binds Chimerin. The invention further relatesto a method of screening for antibodies that specifically bind Chimerincomprising the steps of contacting the unique C-terminal 39 amino acidsof Chimerin (YHITLLPLFEESSKSNANEKHYNLLYTLCFRILAFSIVT) with said testantibody and detecting or measuring whether said test antibody bindssaid Chimerin polypeptide. Further preferred is a method to usecompositions comprising this antibody in diagnostic assays to measureChimerin concentration in bodily fluids, including saliva

Further preferred is a method to use compositions comprising thisantibody to specifically purify Chimerin from bodily fluids, includingsaliva, or from recombinant sources utilizing compositions comprisingthe nucleotide sequence of SEQ NO:5 (Chimerin) or fragments thereof.

Protein of SEQ ID NO:2 (Internal Designation Clone223583_(—)114-044-2-0-E11-F)

The cDNA of clone 223583_(—)114-044-2-0-E11-F (SEQ ID NO:1) encodes theprotein of SEQ ID NO:2 comprising the amino acid sequence:

MAACQLLLEITTFLRETFSCLPRPRTEPLVASTDHTKMPSQMEHAMETMMFTFHKFAGDKGYLTKEDLRVLMEKEFPGFLENQKDPLAVDKIMKDLDQCRDGKVGFQSFFSLIAGLTIACNDYFVVHMKQKGKK. Accordingly it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:2 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone223583_(—)114-044-2-0-E11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:1described throughout the present application also pertain to the nucleicacids included in Clone 223583_(—)114-044-2-0-E11-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:1, SEQ ID NO:2, and Clone 223583_(—)114-044-2-0-E11-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:2 encodes S-100A10 Related Protein(S-100A10rP), which is a splice variant of S-100A. Specifically, theprotein of SEQ ID NO:2 encodes the S-100A10 polypeptide preceded by aunique sequence of 37 amino acids at the amino terminus comprisingMAACQLLLEITTFLRETFSCLPRPRTEP LVASTDHTK.

Dimeric S-100A10 can associate with dimeric annexin II to form aheterotetramer. As a component of this heterotetramer, S-100A10 canmediate a number of activities at the cell surface (Kassam G., et al.,Biochemistry 37:16958-66 (1998), Mai, J., et al., J. Biol. Chem.275:12806-12 (2000) which disclosures are hereby incorporated byreference in their entirety). S-100A10rP antagonizes these activities.

Heterotetrameric annexin II at the cell surface promotes the generationof plasmin, a serine protease with broad substrate specificity, throughits association with both plasminogen and tissue plasminogen activator.The promotion of plasmin generation by annexin II plays a role in: (i)control of hemostasis and coagulation, (ii) macrophage migration andmatrix remodeling,(iii) neuronal cell differentiation, (iv) tumor cellinvasion and metastasis, and (v) cardiovascular development andangiogenesis.

A preferred embodiment of the invention is directed to compositionscomprising the amino acid sequence of SEQ NO:2 (S-100ArP). Furtherpreferred embodiment of the invention is directed to compositionscomprising either monomeric or dimeric S-100A10rP. Further included inthe invention are fragments of the full-length S-100A10rP polypeptidehaving a biological activity described herein as well as thepolynucleotides encoding these fragments.

Further preferred is a method to use compositions comprising S-100ArP tosuppress plasmin generation and thereby decrease inflammation at sitesof chronic inflammation, preferably in the synovium of a joint capsule.Said methods comprise contacting S-100A10rP compositions with thesynovial fluid of the joint capsule. Preferred delivery of S-100A10rPincludes injection or transdermal contact at the site of the joint.

Preferred is a method to use compositions comprising S-100ArP tosuppress tumor cell metastasis. Further preferred is an embodiment ofthe method directed to the use of compositions of S-100A10rP to suppresstumor cell metastasis facilitated by the binding of the cysteineprotease cathepsin B to cell surface hetertetrameric annexin II. Saidmethod is comprised of contacting said tumor cells with an effectivedose of S-100A10rP by injection. Further preferred is an embodiment ofthe method directed to the use of S-100A10rP to suppress the metastasisof breast cancer. Further preferred in an embodiment of the methoddirected to the use of S-100A10rP to suppress the metastasis of glioma.

Preferred is a method to use compositions comprising S-100ArP tosuppress inflammation associated with wound healing. Further preferredare compositions comprised S-100ArP used in methods of treatmentcomprised of contacting a wound or injured tissue with an ameliorativeeffective amount by injection or transdermal contact at the site of thewound.

Acute promyelocytic leukemia (APL) is characterized byhyperfinbrinolysis due to heterotetrameric a unexin II promoted plasmingeneration and a consequential disseminated intravascular coagulation.In a preferred embodiment of the invention, S-100A10rP is used tosuppress this hyperfibrinolysis. Said method is comprised of contactingAPL cells with an effective amount of S-100A10rP by injection.

A preferred embodiment of the invention is to use compositionscomprising S-100A10rP in a method to suppress angiogenesis associatedwith the growth of solid tumors. Further preferred is a method to usecompositions comprising S-100A10rP to suppress angiogenesis associatedwith breast cancer, prostate cancer, pancreatic adenocarcinoma,colorectal cancer, renal cell carcinoma, squamous cell carcinoma of thelung, and T cell lymphoma. Preferred delivery includes contacting thetumor with an effective amount of S-100A10rP by intravenous injection.

A preferred embodiment of the invention is to use compositionscomprising S-100A10rP in a method to suppress angiogenesis associatedwith chronic inflammation. Further preferred is a method to usecompositions comprising S-100A10rP to suppress angiogenesis associatedwith rheumatoid arthritis and thereby decrease inflammation, preferablyin the synovium of a joint capsule. Said methods comprise contactingS-100A10rP compositions with the synovial fluid of the joint capsule.

In a further preferred embodiment, the present invention provides for anantibody that specifically binds an S-100A10rP polypeptide of thepresent invention in a method of neutralizing S-100A10rP function andthereby up-regulating the functional activity of extracellularheterotetrameric annexin II. Further preferred is a method to usecompositions comprising this antibody to promote angiogenesis inischemic heart tissue. Preferred delivery includes contacting the hearttissue with an effective amount of anti-S-100A10rP antibody byintravenous injection. Further preferred is a method to use compositionscomprising anti-S-100A10rP antibody to promote neuritogenesis inischemic brain tissue. Preferred delivery includes contacting the neuraltissue with an effective amount of anti-S-100A10rP antibody by localinjection or transdermal contact.

Protein of SEQ ID NO:32 (Internal Designation clone477709_(—)174-8-2-0-C10-F)

The cDNA of Clone 477709_(—)174-8-2-0-C10-F (SEQ ID NO:31) encodes theprotein of SEQ ID NO:32 comprising the amino acid sequence:

MAWRGWAQRGWGCGQAWGASVGGRSCEELTAVLTPPQLLGRRFNFFIQQKCGFRKAPRKVEPRRSDPGTSGEAYKRSALIPPVEETVFYPSPYPIRSLIKPLFFTVGFTGCAFGSAAIWQYESLKSRVQSYFDGIKADWLDSIRPQKEGDFRKEINKWWNNLSDGQRTVTGIIAANVLVFCLWRVPSLQRTMIRYFTSNPASKVLCSPMLLSTFSHFSLFHMAANMYVLWSFSSSIVNILGQEQFMAVYLSAGVISNFVSYVGKVATGRYGPSLGAALKAIIAMDTAGMILGWKFFDHAAHLGGALFGIWYVTYGHELIWKNREPLVKIWHEIRTNGPKKGGGSK. Accordingly, it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:32 described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in Clone477709_(—)174-8-2-0-C10-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:31described throughout the present application also pertain to the nucleicacids included in Clone 477709_(—)174-8-2-0-C10-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:31, SEQ ID NO:32, and Clone 477709_(—)174-8-2-0-C10-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:32 encodes Pretactilin, a splice variant of theprotein of EMBL entry Q9H300. The corresponding locus located onchromosome 3 possesses at least 2 known variants described in entriesAAH03653 and Q9H300 in EMBL. The closest known sequence, both at thenucleotide and amino acid levels, is Q9H300. Q9H300 is split into 10exons, of which the protein of the invention is missing exon 8, while inAAH03653, it is exon 6 that is absent

Pretactilin is a polypeptide that interacts with the carboxyl-terminusof presenilin-1 and presenilin-2. Pretactilin harbours six putativetransmembrane domains and belongs to the family of transmembranerhomboid like proteins that have been isolated from various organisms,ranging from bacteria, plants, invertebrates to humans. The firstisolated member of this family, the Drosophila melanogaster Rhomboidprotein, is a seven transmembrane domain protein that has beenimplicated in Epidermal Growth Factor Receptor (EGFR) signaling, whichas in mammals controls many aspects of growth and development. Geneticevidence indicates that Rhomboid controls the activation by proteolysisof the transmembrane EGFR ligand, Spitz, a TGFα-like molecule presentsat the surface of neighbouring cells, to generate an active diffusibleform of the ligand.

The rhomboid domain of the Pretactilin extends from amino acid positions186 to 323, and includes the predicted transmembrane domain region. Ithas been recently proposed by Pellegrini et al., 2001, J. AlzheimersDis. 3 (2) which disclosure is hereby incorporated by reference in itsentirety, that the members of the Rhomboid superfamily possess ametal-dependent protease activity.

The familial Alzheimer disease gene products, presenilin-1 andpresenilin-2, are multipass membrane proteins consisting of 6-8 spanningregions that undergo endoproteolytic processing within their largehydrophilic loop at their carboxyl terminus. Immunolocalization studieshave demonstrated that these ubiquitously expressed molecules, primarilylocated to the endoplasmic reticulum and the golgi apparatus, are alsofound on nuclear and plasma membranes. The presenilin proteins have beenreported to be functionally involved in amyloid precursor proteinprocessing, notch receptor signalling, and programmed cell death, orapoptosis.

Alzheimer's Disease (AD) is a devastating neurodegenerative disordercharacterized by progressive memory and cognition impairment associatedwith an increase secretion and deposition of a 4 kDa beta amyloidpeptide (A beta) in extracellular senile plaques in the brain. In bothhealthy and AD patients, A beta is derived by proteolytic cleavage fromthe single transmembrane amyloid precursor protein (APP) by variousproteinases that have been called APP secretases. Alpha secretasescleave APP within the amyloid sequences, whereas other proteases calledbeta- and gamma-secretases cleave on the N- and C-terminal ends,respectively. While a transmembrane aspartyl protease, BACE, has beenidentified as beta-secretase and several proteases may bealpha-secretases (ADAM-10, TACE, PC7), the nature of thegamma-secretase(s) remains elusive. Recently, a number of studies havesuggested that the presenilins themselves, missense mutations in whichcause the most aggressive forms of familial AD with increased productionof A beta, could be the long sought gamma-secretases which releaseA-beta.

The presenilins family of proteins has also been shown to interact withthe Notch signalling pathway by forming stable complexes with Notch andbeing required for its proper cleavage at the cell surface. Notch is asingle transmembrane domain cell surface receptor that mediates manycell fate decisions during development in both vertebrates andinvertebrates. Notch is synthesized as a large precursor that is cleavedin the trans-golgi network lumen to generate two fragments that form aheterodimeric receptor at the cell surface. Following ligand receptorbinding, the C-terminal transmembrane-intracellular fragment of Notch iscleaved within its transmembrane domain by an as yet unidentifiedprotease. This ligand-activated cleavage releases the Notchintracellular domain from the membrane, allowing it to translocate tothe nucleus where it affects the transcriptional activity of targetgenes through interactions with proteins that include members of the CSLfamily.

In addition to their roles in APP processing and Notch receptorsignaling, extensive evidence suggests that presenilins are alsoinvolved in programmed cell death. Over-expression of Presenilin-2increases apoptosis induced by a number of apoptotic stimuli, whereasmutations in the presenilin genes as found in Familial Azheimer'sDisease cases generate molecules with constitutive pro-apoptoticactivity. Complementary studies have demonstrated that depletion of PS2protein levels by antisense RNA protects cells against apoptosis inducedby a number of cell-death-inducing apoptotic stimuli. At the molecularlevel, it has been observed recently that the carboxyl-termini ofpresenilin-1 and presenilin-2 interact with Bcl-XL protein, ananti-apoptotic member of the Bcl-2 family, providing an additional linkbetween these proteins and the apoptotic pathway.

By virtue of its being either a transmembrane protease or atransmembrane protease cofactor, Pretactilin interacts physically withpresenilins to form active complexes in the membranes that are involvedin APP metabolism, Notch signalling and programmed cell death viaspecific protein processing. Specifically, Pretactilin contributes tothe proteolytic processing of a number of protein substrates includingAPP and Notch.

In one embodiment of the present invention, Pretactilin can be used in aprotease cocktail in order to digest proteins, preferentiallytransmembrane proteins, from a biological sample. Use of a proteasecocktail could be of particular interest either to quickly purify DNAfrom crude cellular extracts or to remove transmembrane andmembrane-associated proteins in isolated membranes preparation in orderto prepare protein-free membranes vesicles useful for proteinreconstitution and functional assays in vitro. In a preferredembodiment, Pretactilin is added to a protease cocktail in combinationwith one or more presenilin proteins.

In another embodiment, Pretactilin can be used as a transmembrane markerthat would be useful during protein purification methods for monitoringthe recovery of transmembrane proteins from a biological sample or fromcells grown in vitro. In such methods, the proteins can be detected inany of a number of ways. For example, Pretactilin can be labeled andadded to the sample or the cells prior to the purification step.Alternatively, Pretactilin can be recombinantly fused to a detectableprotein such as GFP and expressed in the organism from which the samplewill be taken, or in the cells, prior to purification. In addition,Pretactilin can be detected throughout the purification steps using amonoclonal or polyclonal antibody that specifically recognizesPretactilin.

The present invention also provides new methods to purify wild type andmutant presenilin proteins, preferentially human presenilins, consistingin using Pretactilin or fragments thereof to co-immunopurify presenilinsfrom cellular extracts. Methods to co-immunopurify proteins are wellknown to those skilled in the art. For example, presenilins can beco-immunopurified by affinity column chromatography or by immobilisationon sepharose-beads with monoclonal or a polyclonal antibody thatspecifically binds Pretactilin. Such purified wild type and mutantpresenilins would then be of particular interest to generate presenilinantibodies that could be used for the treatment of Alzheimer's disease.In addition, the purified presenilin polypeptides could subsequently beused for the diagnosis of Alzheimer's disease as described below.

In a further embodiment, the present invention is used in a diagnosticmethod for detecting Alzheimer's disease in an individual comprising thesteps of:

-   -   (a) co-immunopurifying presenilins with Pretactilin from a        biological sample,    -   (b) adding the corresponding purified polypeptides to membranes        vesicles containing a reconstituted presenilins substrate,        preferentially the Notch protein, as well as, optionally, a        reconstituted Pretactilin,    -   (c) quantifying protease activity of these membrane vesicles        compared to reconstituted positive and negative controls (e.g.,        identical membrane vesicles where wild type and mutant        presenilins have been incorporated, respectively), by        proteolytic fragment detection and quantification.

In another embodiment, the present invention provides new methods toidentify other proteins that interact physically with presenilins and/orPretactilin. In a preferred method, Pretactilin is used toco-immunopurify presenilin complexes from cellular extracts,preferentially from brain cellular extracts, then disrupting theisolated complexes in order to release its components and identifyingthe associated proteins, for example by microsequencing followed by genecloning and characterisation. Alternatively, Pretactilin can be used asbait in two-hybrid experiments in yeast for the screening of interactingpolypeptides. Because such interacting proteins would likely be alsoinvolved in the modulation of A beta peptide production, theircharacterisation would certainly lead to the identification of new geneswhose mutations cause or predispose to Alzheimer's disease. They wouldalso provide useful novel targets for gene and drug therapies of thedisease.

In a further embodiment, Pretactilin can be used in a method to locatepresenilins in subcellular compartments of a cell, preferentiallyneuronal cells, comprising the steps of contacting an isolated sample ofcells with labeled Pretactilin and detecting the labeling in thosecells. Methods used for labeling proteins are well known in the art, anyof which can be used in the present invention.

Pretactilin also provides a method to restore normal APP processing inmutant cells producing increased level of A beta peptide by reducing thelevel or the activity of the present protein in the cells. This can beachieved using techniques well known in the art, for example usingantibodies, antisense molecules, ribozymes, or administrating to saidmutant cells small molecule inhibitors of Pretactilin.

The present invention also provides an in vitro system useful to screenfor inhibitors of A beta production that could be of particular interesteither for the prevention or the treatment of Alzheimer's disease,consisting in transfecting cultured cells in vitro, preferentially braincells, more preferentially neuronal cells, with a nucleotide sequenceencoding Pretactilin placed under the control of a strong constitutivepromoter sequence in order to achieve high expression level ofPretactilin in those cells, applying to the cells the substance to betested, measuring the amount of A beta peptide produced by these cellscompared to control transfected cells.

In another embodiment, Pretactilin can be used to modulate apoptosis ofcells. For example, the level of Pretactilin can be increased in cells,preferentially in tumor cells, in vitro or in vivo, thereby inducingapoptosis. The level or the activity of Pretactilin can be increased inany of a number of ways, including by administering purified Pretactilinto the cells, transfecting the cells with a polynucleotide encodingPretactilin, or administering a compound to the cells that causes anincrease in the activity or expression of Pretactilin. Alternatively,apoptosis can be inhibited by decreasing the level or the activity ofPretactilin in cells, for example using antibodies, antisense molecules,ribozymes, or small molecule inhibitors of Pretactilin. In a preferredembodiment, Pretactilin is used to inhibit apoptosis of neuronal cellsin patients suffering of neurodegenerative diseases, preferentially,Alzheimer's disease.

In another embodiment, the present invention provides a transgenicnon-human animal, preferentially a mammal, more preferentially a rodent,producing high level of A beta peptide due to overproduction ofPretactilin. Such trangenic animal would provide a useful in vivo modelto study the onset of Alzheimer's disease and more particularly toinvestigate the role of A beta 10 peptide deposits in the etiology ofthe disease. It would also be of considerable interest for the screeningof compounds that inhibit A beta peptide secretion or accumulation. Suchtransgenic animal can be obtained by any of the current methods used togenerate transgenic animals that are well known for those skilled in theart, for example in the mouse, using DNA microinjection into fertilizedeggs or transfection of embryonic stem cells. High over-expression ofPretactilin can be achieved by placing the nucleotide sequence encodingPretactilin under the control of a strong promoter sequence. Thepromoter sequence can be derived from a gene having a broad expressionin the animal or from a gene whose expression is restricted to thebrain. Preferentially, a regulatable promoter sequence is used in orderto control temporally the expression of the transgene once introducedinto the animal.

In another embodiment, the level or the activity of Pretactilin can bemodulated to provide a treatment for Alzheimer's disease in a patient.Indeed as A beta peptide deposition is an early and invariant event inAlzheiner's disease, it is believed that a treatment that affects A betaproduction will be useful in the treatment of the disease. Accordingly,reducing level or activity of Pretactilin in mutant cells would therebydiminish A beta production. This could be achieved by any of the wellknown strategies used for therapy in vivo, for example using antisensmolecules, antibody or small molecule inhibitors of Pretactilin.

Protein of SEQ ID NO 34: (Internal Designation145606_(—)106-023-2-0-B3-F):

The cDNA of clone (SEQ ID NO:33) encodes the human MS4A5 protein,comprising the sequence:

MDSSTAHSPVFLVFPPEITASEYESTELSATTFSTQSPLQKLFARKMKILGTIQILFGIMTFSFGVIFLFTLLKPYPRFPFIFLSGYPFWGSVLFINSGAFLIAVKRKTTETLIILSRIMNFLSALGAIAGIILLTFGFILDQNYICGYSHQNSQCKAVTVLFLGILITLMTFSIIELFISLPFSILGCHSEDCD CEQCC(SEQ ID NO:34). Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:34 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in clone145606_(—)106-023-2-0-B3-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:33described throughout the present application also pertain to the nucleicacids included in clone 145606_(—)106-023-2-0-B3-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:33, SEQ ID NO:34, and Clone 145606_(—)106-023-2-0-B3-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The cDNA of SEQ ID NO:33 comprising 5 exons encodes the 200 amino-acidMS4A5 protein (STR Q9H3V2), which belongs to the MS4A protein family(membrane-spanning four-domains, subfamily A). Four members of MS4Afamily in human (MS4A4-7) and in mouse (MS4A8-11) have been described(Ishibashi K. et al, Gene (2001), 264, 87-93 which disclosure is herebyincorporated by reference in its entirety). As with the other members ofthe CD20/Fc(sigma)RI(beta)/HTm4 superfamily, all MS4A proteins arehighly hydrophobic with four transmembrane domains (but are distinctfrom tetraspanin family members which also have four transmembranedomains). The cDNA of SEQ ID NO:33 encoding the protein of SEQ ID NO:34possesses a conserved sequence around the initiating methionine (ATC ATGG) and a consensus protein kinase A (PKA) phosphorylation site (KRKTT)at the intracellular loop between the second and third transmembranedomains. In contrast with other members of MS4A family, which are mostlyexpressed in lymphoid tissues, MS4A5 is expressed in testis, pancreas,and at low levels in the heart and brain. The gene of MS4A5 is locatedon human chromosome 11, specifically at position 11q12, the samechromosome as the CD20, Fc(sigma)RI(beta) and HTm4 genes. MS4A5 is anovel transmembrane protein that acts alone or in combination with otherproteins as an ion channel, e.g. a ligand-gated calcium channel. MS4A5is involved in a number of cellular functions in non-lymphoid cells, forexample intracellular signaling, regulating intracellular calciumconcentrations, exocrine functions, and endocrine functions.

In one embodiment, the protein of the invention or fragment thereofprovides a method to detect cells specifically expressing the presentprotein, using for example flow cytometry technology or classical insitu detection techniques which are well known in the art. Such methodsare useful, e.g. to specifically detect cells of the testis, pancreas,heart, or brain, as the present protein is highly expressed in thesecell types. Such methods are also useful to detect cells over- orunder-expressing the present protein, and is thus useful for diagnosingdiseases or conditions resulting from or associated with an increase ordecrease in expression or activity of the protein. This method includesthe steps of contacting a biological sample obtained from an individualsuspected of suffering from the disease or condition, or at risk ofdeveloping the disease or condition, with a compound capable ofselectively binding the present protein or nucleic acids, e.g. anantibody directed against the present protein or a polynucleotide probedirected against the present cDNA. Following this binding step, themethod further comprises detecting the presence or absence of selectivebinding between the compound and the cells or proteins within thesample. In preferred embodiments, the compound is labeled, and thesample comprises cells derived from the testis, pancreas, heart, orbrain.

In another embodiment, the protein of the invention or fragment thereofcan be used to modulate the proliferation of cells. For example, thelevel or activity of the present protein can be increased in cells toincrease the rate or extent of proliferation of the cells. In one suchembodiment, the proliferation of cells in a biological sample isincreased by contacting the biological sample with an amount of thepresent protein sufficient to increase the rate or extent ofproliferation of one or more cells within the sample, or with a compoundthat increases the activity or expression of the present protein withinone or more cells of the sample. Such methods can be performed either invitro or in vivo and, preferably, the cells comprise pancreatic,testicular, heart or brain cells. The level of the present protein canbe increased in cells in any of a number of ways, including byadministering purified protein to the cells, transfecting the cells witha polynucleotide encoding the protein, or administering a compound tothe cells that causes an increase in the activity or expression of theprotein. Alternatively, proliferation of cells can be inhibited bydecreasing the level of the present protein in cells, for example usingantisense molecules, or more specifically inhibit the activity of thepresent protein using direct or indirect inhibitor molecules orantagonistic antibodies directed against the present protein.

In a further embodiment, the protein of the invention or fragmentthereof can be used to modulate cellular calcium concentration andthereby modulate calcium-dependant signaling. Calcium transport can bemodulated, for example, by contacting a biological sample with an amountof the present protein sufficient to increase calcium transport of oneor more cells within the sample, or with a compound that increases theactivity or expression of the present protein within one or more cellsof the sample. Such methods can be used either in vitro or in vivo andpreferably, but not limited to, the methods are performed on cellscomprising pancreatic, testicular, heart or brain cells. The level ofthe present protein can be increased in cells in any of a number ofways, including by administering purified protein to the cells,transfecting the cells with a polynucleotide encoding the protein, oradministering a compound to the cells that causes an increase in theactivity or expression of the protein. Alternatively, the activity ofthe present protein can be inhibited by decreasing the level of thepresent protein in cells, for example using antisense molecules, byusing direct or indirect inhibitor molecules or antagonistic antibodiesof the present protein, or by expressing in the cells an inactive formof the protein that acts in a dominant negative fashion to inhibit thenormal calcium signalling in the cells carried out by other members ofthe MS4A family.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Such animals represent an in vivo assaymethod for testing candidate molecules potentially useful for thetreatment of various pathophysiological aspects of diseases associatedwith abnormal calcium homeostasis and/or cell growth or any functionspecifically related to the activity of the present protein. Theseanimals can be generated with any method of increasing or decreasing theexpression of the present protein.

In another embodiment, since calcium is an universal intracellularmessenger, controlling a diverse range of cellular processes such asgene transcription, cell proliferation, and more specifically musclecontraction, synaptic function, secretion of insulin in pancreaticislets of Langerhans, and many others, the present protein or fragmentthereof provides a method of treating different pathological statesarising from or associated with destabilization of calcium homeostasisin many organs (brain, kidney, parathyroid gland, pancreas, bone,intestine). In addition, any of these processes can be enhanced orinhibited in cells or in patients, even when the protein is at normallevels in the cells or in the cells of the patient, by causing adecrease or increase in the normal level of the protein in the cells.For any of the herein-described methods, the activity of the presentprotein can be increased or inhibited in any of a large number of ways,for example by using polyclonal or monoclonal antibodies, or any othercompound having qualitative biological activity in common with afull-length antibody, that specifically binds to the present protein andexerts stimulatory or inhibitory effects on functions involving thepresent protein.

Any compound interacting with the present protein and thereby promotingor interfering with its activities can also be used as a method oftreating any of the pathologies described above. Such compounds can beidentified, e.g., using interaction-screening approaches such as, butnot limited to, co-immunoprecipitation, two-hybrid methods. Further,compounds can be screened for the ability to modulate the activity ofthe present protein by providing a cell expressing the present protein,or providing lipid bilayers reconstituted with the present protein, anddetecting the ability of a compound to modulate the activity of thepresent protein in the cell or in the bilayer. Such activity can bedetected in any of a large number of ways, including but not limited todetecting calcium flux or calcium signalling in the cells or membranes,e.g. as manifest in the activity of downstream members of the signaltransduction pathway. The present invention also provides an in vitromethod to identify any compound able to promote or interfere with someor all activities of the present protein, the method comprising thesteps of contacting the present protein with a test compound anddetecting the ability of the compound to bind to or modulate theactivity of the protein. Also in this embodiment, the present protein orany effective compound identified by this way of investigation usefulfor the treatment of disorders described above can be used incombination with other drugs or compounds.

As it has been shown that multiple loci on chromosome 11q13 are relevantto atopic asthma (Adra C N. et al, Clin. Genet. (1999) June;55(6):431-437), the present invention also provides a novel candidategene for this condition. Accordingly, the present invention providesmethods for the diagnosis of atopic asthma, the method comprisingdetermining the identity of one or more nucleotides of the presentnucleic acids in one or more cells of an individual suspected of havingthe condition, or at risk of developing the condition, and determiningif the cell or cell contains a nucleotide within the present nucleicacid sequence indicative of the condition, or of an elevated risk ofdeveloping the condition. The identity of such nucleotides can bedetermined in any of a number of ways, for example using any standardsequencing or genotyping method, many of which are well known in theart.

Protein of SEQ ID NO:36 (Internal Designation Clone1000769575_(—)208-22-1-0-B2-F)

The cDNA of Clone 1000769575_(—)208-22-1-0-B2-F (SEQ ID NO:35) encodesthe protein of SEQ ID NO:36 comprising the amino acid sequence

MGMSSLKLLKYVLFFFNLLFWICGCCILGFGIYLLIHNNFGVLFHNLPSLTLGNVFVIVGSIIMVVAFLGCMGSIKENKCLLMSFFILLLIILLAEVTLAILLFVAKGLTDSIHRYHSDNSTKAAWDSIQSFLQCCGINGTSDWTSGPPASCPSDRKVEGCYAKARLWFHSNFFIRGPY. Accordingly itwill be appreciated that all characteristics and uses of polypeptides ofSEQ ID NO:36 described throughout the present application also pertainto the polypeptides encoded by the nucleic acids included in Clone1000769575_(—)208-22-1-0-B2-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:35described throughout the present application also pertain to the nucleicacids included in Clone 1000769575_(—)208-22-1-0-B2-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:35, SEQ ID NO:36, and Clone 1000769575_(—)208-22-1-0-B2-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:36 encodes Antaginin, a complex splice variantof CD53 with novel function. In Antaginin, splicing of exon 4 onto exon5 results in a deletion of 9 amino acids (4 from exon 4,5 from exon 5)and a correspondingly unique junctional sequence. In addition, splicingof exon 7 onto normally 3′-untranslated nucleotide sequence within exon8 results in a deletion of 14 amino acids from exon 7, as well as thedeletion of the carboxy-terminal 23 amino acids of CD53 and itsreplacement with a unique carboxy-terminal sequence of 6 amino acids inAntaginin.

CD53, restricted in expression to leukocytes, is a member of thetetraspaninin superfamily. CD53 is an integral membrane proteincharacterized by four transmembrane domains (TM1-TM4), forming a smalland a large extracellular loop (EC1 and EC2, respectively), with shortintracellular amino and carboxyl tails. EC1 and EC2 of CD53 comprise theamino acid sequences 37-54 and 107-181, respectively (numbered from theinitiating methionine of CD53). TM1-TM4 of CD53 comprise the amino acidsequences 11-36, 55-69, 81-106, and 182-206, respectively (numbered fromthe initiating methionine of CD53) (Rost, B. et al., Prot. Sci.5:1704-18, (1996) which disclosure is hereby incorporated by referencein its entirety).

CD53 facilitates the assembly of modular signalling complexes at thecell surface. Specifically, CD53 acts as an adaptor to functionally linkan extracellular ligand-binding domain (such as that of beta 1 integrin)to an intracellular domain involved in signal transduction (such as thatof protein kinase C) (Zhang, X A et al., J. Biol. Chem. (2001) whichdisclosure is hereby incorporated by reference in its entirety). Beta 1integrin has been shown to associate with CD53 through EC2. Moreover,through its interaction with other tetraspaninins, CD53 is incorporatedinto a higher order tetraspaninin web exisiting at the cell surface.CD53 displays numerous properties that indicate its physiologicalimportance in cell adhesion, motility, activation (including thedelivery of a co-stimulatory signal to for CD3/T cell receptor-mediatedT cell activation), and proliferation (Boucheix, C. et al. ExpertReviews in Molecular Medicine (2001) which disclosure is herebyincorporated by reference in its entirety).

Antaginin is characterized by a highly perturbed EC2 loop and a highlydivergent TM4 transmembrane domain. The EC2/TM4 region of Antaginincomprises amino acids 107-179 (numbered from the initiating methionineof Antaginin). In addition, Antaginin is characterized by anextracelluar perturbation of the amino acid sequence at the junction ofexons 4 and 5 (amino acids 124/125, numbered from the initiatingmethionine of Antaginin) (Rost, B. et al., Prot. Sci. 5:1704-18, (1996)which disclosure is hereby incorporated by reference in its entirety).Antaginin antagonizes CD53-facilitated assembly of functional modularsignalling complexes at the cell surface.

In a preferred embodiment, the present invention provides for anantibody that specifically binds Anataginin of the present invention.Further preferred is a method for making such antibody wherein a mouseis immunized with a syngeneic cell line transfected with Antaginin.Monoclonal antibodies derived from said mouse are screened for bindingto the Antaginin-transfected cell line but not to the identical cellline transfected with human CD53. Antibody specificity is furtherestablished through amino acid sequence analysis of immunoprecipitatedmaterial. Further preferred is a method for making said antibody whereinsaid antibody binds to EC1 or the sequence carboxyl-terminal (EC2/TM-4region) of Antaginin. EC1 and the EC2/TM4 region of Antaginin comprisethe amino acid sequences 37-54 and 107-179, respectively (numbered fromthe initiating methionine of Antaginin). Further preferred is a methodfor making said antibody wherein said antibody binds to the EC2/TM4region of Antaginin. Methods of generating said monoclonal antibody andof establishing its specificity are well known to those skilled in theart.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Antaginin.Further preferred is a method for using said antibody diagnostically todetermine the basis for an impaired immune response. Further preferredis a method of using said antibody diagnostically in a flow cytometricanalysis of Antaginin expression by leukocytes in a pathologicalcontext. Further preferred is a method of using said antibodydiagnostically in a flow cytometric analysis of Antaginin expression byleukocytes in the context of viral infection wherein the virus isselected from, but not restricted to, the group consisting of: (a)Cytomegalovirus; (b) Human immunodeficiency virus; (c) Human herpesvirus 6 (HHV 6); (d) Hepatitis C virus; and (e) Hepatitis D virus.

Further preferred is a method of using said antibody diagnostically in aflow cytometric analysis of Antaginin expression by normal leukocytes inthe leukemic patient to determine the basis for an impaired anti-tumorimmune response wherein the leukemia is selected from, but notrestricted to, the group consisting of: (a) B cell acute lymphoblasticleukemia (B-ALL); (b) Chronic lymphocytic leukemia (CLL); (c) T cellacute lymphoblastic leukemia (T-ALL); (d) Multiple myeloma; and (e)Acute myeloid leukemia (AML).

Further preferred is a method of using said antibody diagnostically in aflow cytometric analysis of Antaginin expression by normal leukocytes inthe cancer patient to determine the basis for an impaired anti-tumorimmune response wherein the cancer is selected from, but not restrictedto, the group consisting of. (a) Melanoma; (b) Breast carcinoma; (c)Lung carcinoma; (d) Colon carcinoma; (e) Hodgkin's lymphoma; (f)Non-Hodgkin's lymphoma; (g) Prostatic carcinoma; (h) Pancreaticcarcinoma; (i) Uterine carcinoma; (j) Ovarian carcinoma; (k) Testicularcarcinoma; (l) Renal carcinoma; (m) Hepatic carcinoma; and (n) Lungnon-small-cell carcinoma.

The threshold for leukocyte activation can be regulated by cytokine. Ina further embodiment, the present invention provides for the use of saidAntaginin antibody in in vitro analysis of cytokine regulation ofAntaginin expression by leukocytes. Further preferred is a method ofusing said antibody in a flow cytometric analysis of said regulation bycytokine wherein the cytokine is selected from, but not restricted to,the group consisting of: (a) Interferon gamma; (b) Interleukin 17; (c)Interleukin 4; (d) Interleukin 10; (e) Interleukin 13; (f) Interleukin15; (g) Interleukin 1; (h) Interleukin 6; (i) Monocyte chemotacticprotein 1 (MCP-1); 0) Interleukin 8; and (k) Tumor necrosis factoralpha.

Further preferred is a method of contacting said antibody with Antagininand thereby sterically inhibiting the capacity of Antaginin toantagonize the CD53-facilitated assembly of functional modularsignalling complexes at the cell surface. In so doing, said Antagininantibody up-regulates CD53-mediated leukocyte activation. Preferredcompositions comprise the Antaginin antibody or fragments or derivativesthereof. Preferred route of administration is intravenous injection.

In a further embodiment of the invention, said Antaginin antibody isincorporated as an adjuvant in vaccine preparations in a method toup-regulate the elicited immune response. In said method, said Antagininantibody facilitates the CD53-mediated leukocyte activation contributingto establishment of specific immunity. Said Antaginin antibodyup-regulates CD53-mediated leukocyte activation by sterically inhibitingthe capacity of Antaginin to antagonize the CD53-facilitated assembly offunctional modular signaling complexes at the cell surface. Furtherpreferred is a method to use said antibody in a vaccine targeting aviral infection wherein the virus is selected from, but not restrictedto, the group consisting of: (a) Human immunodeficiency virus; (b) Humanherpes virus 6 (HHV 6); (c) Hepatitis C virus; (d) Hepatitis D virus;(e) Hepatitis E virus; (f) Cytomegalovirus; (g) Respiratory syncytialvirus; (h) Herpes simplex virus type I; (i) Herpes simplex virus typeII; (j) Influenza virus; (k) Parvovirus; (l) Coxsachie virus; (m)Echovirus; (n) Epstein-Barr virus; (o) Dengue virus; (p) Lassa fevervirus; and (q) Ebola virus.

Further preferred is a method to use said Antaginin antibody in avaccine targeting a protozoan infection wherein the protozoa is selectedfrom, but not restricted to, the group consisting of: (a) Entamoebahistolytica; (b) Cryptosporidium parvum; (c) Plasmodium falciparum; (d)Trypanosoma; (e) Leishmania; (f) Trichomonas vaginalis; and (g)Acanthamoeba.

Viruses can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Antagininantibody is used in a method of up-regulating the immune responseagainst an ongoing viral infection. In said method, said Antagininantibody facilitates the CD53-mediated leukocyte activation contributingto the anti-viral immune response. Said Antaginin antibody up-regulatesCD53-mediated leukocyte activation by sterically inhibiting the capacityof Antaginin to antagonize the CD53-facilitated assembly of functionalmodular signaling complexes at the cell surface. Further preferred is amethod of up-regulating the immune response against an ongoing viralinfection wherein the virus is selected from, but not restricted to, thegroup consisting of: (a) Human immunodeficiency virus; (b) Human herpesvirus 6 (HHV 6); (c) Hepatitis B virus; (d) Hepatitis C virus; (e)Hepatitis D virus; (f) Cytomegalovirus; (g) Respiratory syncytial virus;(h) Influenza virus; (i) Herpes simplex virus type I; (j) Herpes simlexvirus type II; (k) Epstein Barr virus; (l) Varicella zoster virus; (m)Morbillivirus; (n) Parmyxovirus; (o) Papilloma virus; (p) Adenovirus;(q) Dengue virus; (r) Lassa fever virus; (s) Coxsachie virus; (t)Echovirus; and (u) Ebola virus.

Bacteria can suppress the immune response as a means of evading immunesurveillance. 20 In a further embodiment of the invention, saidAntaginin antibody is used in a method of up-regulating the immuneresponse against an ongoing bacterial infection. In said method, saidAntaginin antibody facilitates the CD53-mediated leukocyte activationcontributing to the anti-bacterial immune response. Said Antagininantibody up-regulates CD53-mediated leukocyte activation by stericallyinhibiting the capacity of Antaginin to antagonize the CD53-facilitatedassembly of functional modular signaling complexes at the cell surface.Further preferred is a method of up-regulating the immune responseagainst an ongoing bacterial infection wherein the bacteria is selectedfrom, but not restricted to, the group consisting of: (a) Mycobacteriumavium complex; (b) Pneumocystis carinii; (c) Acne vulgaris; (d)Legionella pneumophilia; (e) Yersinia pestis; (f) Ureaplasmaurealyticum; (g) Chlamydia pneumoniae; (h) Helicobacter pylori; (i)Treponema pallidum; (j) Neisseria gonorrhoeae; (k) Salmonellatyphimurium; (l) Vibrio cholera; (m) Clostridium difficile; (n)Bacillary dysentary; (o) Pencillin resistant Pneumococcus; (p)Burkholderia mallei; (q) Mycobacterium leprae; (r) Mycobacteriumhaemophilum; (s) Mycobacterium kansasii; (t) Haemophilus influenzae; and(u) Bacillus anthracis.

Protozoa can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Antagininantibody is used in a method of up-regulating the immune responseagainst an ongoing protozoan infection. In said method, said Antagininantibody facilitates the CD53-mediated leukocyte activation contributingto the anti-protozoan immune response. Said Antaginin antibodyup-regulates CD53-mediated leukocyte activation by sterically inhibitingthe capacity of Antaginin to antagonize the CD53-facilitated assembly offunctional modular signaling complexes at the cell surface. Furtherpreferred is a method of up-regulating the immune response against anongoing protozoan infection wherein the protozoa is selected from, butnot restricted to, the group consisting of: (a) Entamoeba histolytica;(b) Cryptosporidium parvum (c) Giardia lamblia; (d) Toxoplasma gondii;(e) Isospora belli; (f) Encephalitozoon cuniculi; (g) Enterocytozoonbieneusi; (h) Plasmodium falciparum; (i) Trypanosoma; (j) Leishmania;(k) Trichomonas vaginalis; and (l) Acanthamoeba.

In a further embodiment of the invention, said Antaginin antibody isused in a method of up-regulating the immune response against an ongoingfungal infection wherein the fungus is selected from, but not restrictedto, the group consisting of: (a) Cryptococcal meningitis; (b)Histoplasma capstulatum; (c) Coccidiodes immitis; and (d) Candidaalbicans.

Tumors can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Antagininantibody is used in a method of up-regulating the immune responseagainst a tumor. In said method, said Antaginin antibody facilitates theCD53-mediated leukocyte activation contributing to the anti-tumor immuneresponse. Said Antaginin antibody up-regulates CD53-mediated leukocyteactivation by sterically inhibiting the capacity of Antaginin toantagonize the CD53-facilitated assembly of functional modular signalingcomplexes at the cell surface. Further preferred is a method ofup-regulating the immune response against a tumor wherein the tumor isselected from, but not restricted to, the group consisting of: (a)Melanoma; (b) Breast carcinoma; (c) Lung carcinoma; (d) Colon carcinoma;(e) Hodgkin's lymphoma; (f) Non-Hodgkin's lymphoma; (g) Prostaticcarcinoma; (h) Pancreatic carcinoma; (i) Uterine carcinoma; (j) Ovariancarcinoma; k) Testicular carcinoma; (l) Renal carcinoma; (m) Hepaticcarcinoma; and (n) Lung non-small-cell carcinoma.

In a further embodiment of the invention, said Antaginin antibody isincorporated as an adjuvant in therapeutic anti-tumor vaccines whereinthe tumor is selected from, but not restricted to, the group consistingof: (a) Melanoma; (b) Breast carcinoma; (c) Lung carcinoma; (d) Coloncarcinoma; (e) Hodgkin's lymphoma; (f) Non-Hodgkin's lymphoma; (g)Prostatic carcinoma; (h) Pancreatic carcinoma; (i) Uterine carcinoma;(j) Ovarian carcinoma; (k) Testicular carcinoma; (l) Renal carcinoma;(m) Hepatic carcinoma; and (n) Lung non-small-cell carcinoma.

Intracellular (macrophage) pathogens can be eliminated either throughmacrophage activation or through lysis of infected macrophages bycytolytic T lymphocytes (Chun et al., J. Exp. Med. 193:1213 (2001) whichdisclosure is hereby incorporated by reference in its entirety). In afurther embodiment of the invention, said Antaginin antibody is used ina method to eliminate intracellular pathogens by facilitating macrophageactivation or cytolytic T lymphocyte generation wherein the pathogen isselected from, but not restricted to, the group of intracellular(macrophage) pathogens consisting of: (a) Histoplasma capsulatum; (b)Mycobacterium tuberculosis; (c) Salmonella typhimurium; (d) Chlamydiatrachomatis; and (e) Pneumocystis carinii.

There have been several examples of tetraspanins playing a role in theviral life cycle. Anti-etraspanin antibodies inhibit syncytium formationand/or virus production. This was observed for the tetraspanins CD81 andCD82 with human T-lymphotropic virus 1, and for the tetraspanin CD9 withthe feline immunodeficiency virus and the canine distemper virus. It isalso believed that the tetraspanin CD81 also plays a role in theaetiopathogenesis of hepatitis C virus (Boucheix, C. et al. (2001) whichdisclosure is hereby incorporated by reference in its entirety). In afurther embodiment of the invention, said Antaginin antibody is used ina method of blocking viral infection when Antaginin is used as a virusreceptor. Further preferred is the use of said Antaginin antibody in amethod of blocking said viral infection when Antaginin used as saidvirus receptor and is expressed by a leukocyte type selected from, butnot restricted to, the group of leukocyte types consisting of: (a) Tlymphocyte; (b) B lymphocyte; (c) NK lymphocyte; (d) Monocyte; (e)Macrophage; (f) Neutrophil; and (g) Dendritic cell.

In a further preferred embodiment, the present invention provides for amethod of screening test compounds for the ability to bind Antaginin andeither inhibit or promote the capacity of Antaginin to interfere withCD53 function. Further preferred is a method of screening said testcompounds for the ability to bind Antaginin and either inhibit orpromote the capacity of Antaginin to interfere with CD53 function as itrelates its facilitation of signal transduction through beta 1 integrin(Zhang, X A et al., J. Biol. Chem. (2001) which disclosure is herebyincorporated by reference in its entirety). Further preferred is amethod of screening said test compounds for the ability to bindAntaginin and either inhibit or promote the capacity of Antaginin tointerfere with the CD53-facilitated association of protein kinase C withbeta 1 integrin. Further preferred is a method of screening said testcompounds for the ability to bind Antaginin and either inhibit orpromote the association of protein kinase C with beta 1 integrin in abeta 1 (alpha3beta1, alpha4beta1, or alpha6beta1)-expressing cell linetransfected with CD53 and Antaginin but not in the identical cell linetransfected with CD53 alone. Methods of screening said test compoundsand for characterizing their effect on CD53-facilitated association ofprotein kinase C with beta 1 integrin are well known to those skilled inthe art.

Preferred formulation of said compound is that selected from, but notrestricted to, formulations compatible with the routes of deliveryselected from the group: (a) Oral; (b) Transdermal; (c) Injection; (d)Buccal; and (d) Aerosol.

Compounds found to bind Antaginin and to inhibit the capacity ofAntaginin to interfere with CD53 function, thereby effectivelyup-regulating CD53 activity, are used in methods analogous to thosedescribed above for Antaginin antibody.

Compounds found to bind Antaginin and to promote the capacity ofAntaginin to interfere with CD53 function effectively down-regulate CD53activity. Such compounds have application to chronic inflammatoryautoimmune disease and to other disorders of immune dysregulation. Suchcompounds down-regulate CD53-mediated leukocyte activation by promotingthe capacity of Antaginin to antagonize the CD53-facilitated assembly offunctional modular signaling complexes at the cell surface. In a furtherembodiment of the invention, said compound is used in a method ofcontacting Antaginin to down-regulate a dysregulated immune response andthereby treat the associated immune disorder wherein said immunedisorder is selected from, but not restricted to, the group: (a)Rheumatoid arthritis; (b) Inflammatory bowel disease; (c) Insulindependent diabetes mellitus (Type 1 diabetes); (d) Multiple sclerosis;(e) Systemic lupus erythematosus; (f) Psoriasis; (g) Allergic asthma;(h) Allergic rhinitis (hayfever); and (i) Graft versus host disease. Ina further embodiment of the invention, said test compound having theability to promote the capacity of Antaginin to interfere with CD53function is used in a method to suppress acute inflammation. Said testcompounds down-regulate CD53-mediated leukocyte activation by promotingthe capacity of Antaginin to antagonize the CD53-facilitated assembly offunctional modular signalling complexes at the cell surface. Furtherpreferred is a method to use said test compound to suppress inflammationassociated with wound healing. Further preferred are compositionscomprised of said test compound used in methods of contacting a wound orinjured tissue with an ameliorative effective amount by injection ortransdermal contact at the site of the wound.

Protein of SEQ ID NO:38 (Internal Designation Clone146994_(—)106-023-4-0-C9-F)

The cDNA of Clone 146994_(—)106-023-4-0-C9-F (SEQ ID NO:37) encodes theprotein of SEQ ID NO:38 comprising the amino acid sequence:

MSPGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA. Accordingly it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:38 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone146994_(—)106-023-4-0-C9-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:37described throughout the present application also pertain to the nucleicacids included in Clone 146994_(—)106-023-4-0-C9-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:37, SEQ ID NO:38, and Clone 146994_(—)106-023-4-0-C9-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:38 encodes Beferin. Beferin is a novel splicevariant of two recently described members of the B lymphocyte activationantigen B7 (BLAA) family, B7-H3 and Blaa. Beferin has novel function asdescribed below.

B7-H3 was identified as a human B7-like molecule with T lymphocytecostimulatory activity (Chapoval, A I et al., Nature Immunology 2:269-74(2001) which disclosure is hereby incorporated by reference in itsentirety). B7-H3 has the structure:

[Signal peptide]-[IgV-ike domain 1]-[IgC-like domain 2]-[transmembraneregion]-[cytoplasmic tail].

Blaa (NCBI Accession No. AX097550) was identified as a human B7-likemolecule, as described in Patent Application WO00118204A1(“Polynucleotides encoding members of the human B lymphocyte activationantigen B7 family and polypeptides encoded thereby”) and incorporated byreference in its entirety. Blaa has the structure:

[Signal peptide]-[IgV-like domain 1]-[IgC-like domain 1]-[IgV-likedomain 2]-[IgC-like domain 2]-[transmembrane region]-[cytoplasmic tail].

Blaa (NCBI Accession No. AX047070) was independently identified as aprotein with beta-secretase (beta-amyloid-converting enzyme) activity,as described in Patent Application WO00068266A1 (“Amyloid precursorprotein protease and related nucleic acid compositions”) andincorporated by reference in its entirety. The amino acid sequence ofAX947070 is identical to that of AX097550.

IgV-like domain 1 is highly similar, but not identical, to the aminoacid sequence of IgV-like domain 2. IgC-like domain 1 is highly similar,but not identical, to the amino acid sequence of IgC-like domain 2.

In the case of Beferin, a novel 5′ exon is spliced directly onto theexons encoding the transmembrane region and cytoplasmic tail. Thisresults in the deletion of the IgV-like and IgC-like extracellulardomains. The short extracellular tail of Beferin is comprised ofapproximately seven amino acids shared with B7-H3 and Blaa preceded bythree novel (not found in either B7-H3 or Blaa) N-terminal amino acidsencoded by the novel 5′ exon (underlined here): MSPGQPMTFP.

Costimulation, in addition to T cell receptor engagement, is requiredfor optimal activation of T cells. The most extensively studiedcostimulatory molecules are members of the B lymphocyte activationantigen B7 family, of which there are presently five. Each B7 familymember binds to one or more counter-receptor on the T cell, of whichthere are presently four. B7-H3 is highly expressed in many humantissues including heart, liver, placenta, prostate, testis, uterus,pancreas, small intestine, and colon. Low expression of B7-H3 was alsofound in brain, skeletal muscle, kidney, and lung. B7-H3 is notdetectable in peripheral blood mononuclear cells, although it can beinduced on dendritic cells and monocytes by inflammatory cytokines.Several tumor lines also express B7-H3, including those derived frommelanoma, cervical adenocarcinoma, chronic myelogenous leukemia, lungcarcinoma, and colorectal adenocarcinoma. B7-H3 costimulatesproliferation of both CD4⁺ and CD8⁺ T cells, enhances the induction ofcytotoxic T lymphocytes (CTL), and selectively stimulatesproinflammatory cytokine interferon gamma (IFNgamma) production in thepresence of T cell receptor signaling. B7-H3 exists as non-covalentoligomers on the antigen-presenting cell, and this is important forhigh-avidity binding of B7-H3 to its counter-receptor in its role as Tcell costimulator.

In non-neuronal tissue, Blaa cleaves the 751 amino acid isoform ofamyloid beta protein precursor (APP751) at the cell surface by virtue ofits beta-secretase activity to generate a soluble fragment identical tothe serine protease inhibitor protease nexin 2 (PN2). PN2 and its Kunitzprotease inhibitory domain have been shown to be inhibitors ofcoagulation factor VIIa (FVIIa) and factor VIIa-tissue factor complex(FVIIa-TF) (Mahdi, F et al., Thromb. Res. 99:267-76 (2000) whichdisclosure is hereby incorporated by reference in its entirety)initiators of the extrinsic coagulation cascade. TF expression and itsengagement of the extrinsic coagulation pathway by ovarian cancer cellshas been shown to play role in metastasis of the cancer (Fischer, E G etal., J. Clin. Invest. 104:1213-21 (1999) which disclosure is herebyincorporated by reference in its entirety). Factor Xa (FXa) generated byFVIIa-TF has been shown to lead to pro-inflammatory activation ofvascular endothelial cells through its cleavage of protease-activatedreceptor 2 (PAR2) (Camerer, E et al., Proc. Natl. Acad. Sci. USA97:5255-60 (2000) which disclosure is hereby incorporated by referencein its entirety). FXa can also elicit a pro-inflammatory cellularresponse by cleavage of protease-activated receptor 1 (PAR 1)(Kravchenko, R M Blood 97:3109-16 (2001) which disclosure is herebyincorporated by reference in its entirety).

Beferin interferes with B7-H3 co-stimulation of T lymphocytes throughits non-productive incorporation into B7-H3 oligomers at the cellsurface. One function of Beferin therefore is to negatively regulate Tlymphocyte co-stimulation. In a pathological context, Beferinup-regulation facilitates evasion of immune surveillance by pathogensand tumor cells.

Beferin interferes with Blaa generation of PN2 through itsnon-productive interactions with APP751. A second functional consequenceof Beferin expression is therefore up-regulated engagement of theextrinsic coagulation coagulation pathway, including the generation ofFXa. In a pathological context, Beferin up-regulation facilitateshypercoagulability and cancer metastasis.

In a preferred embodiment, the present invention provides for anantibody that specifically binds Beferin of the present invention.Further preferred is a method for making such antibody wherein a mouseis immunized with a syngeneic cell line transfected with Beferin.Monoclonal antibodies derived from said mouse are screened for bindingto the Beferin-transfected cell line but not to the identical cell linetransfected with human B7-H3 or Blaa. Antibody specificity is furtherestablished through amino acid sequence analysis of immunoprecipitatedmaterial. Further preferred is a method for making said antibody whereinsaid antibody specifically binds all or in part to the extracellularamino terminus of Beferin. The extracellular amino terminus of Beferinis comprises the amino acid sequence 1-10 (numbered from the initiatingmethionine of Beferin). Methods of generating said monoclonal antibodyand of establishing its specificity are well known to those skilled inthe art.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Beferin.Further preferred is a method for using said antibody diagnostically todetermine the basis for an impaired immune response or forhypercoagulability. Further preferred is a method of using said antibodydiagnostically in a flow cytometric analysis of Beferin expression byleukocytes in a pathological context. Further preferred is a method ofusing said antibody diagnostically in an immunohistochemical analysis ofBeferin expression by tissue in a pathological context. Methods ofcarrying out immunohistochemical or flow cytometric analysis are wellknown to those skilled in the art.

Further preferred is a method of using said antibody diagnostically in aflow cytometric analysis of Beferin expression by normal leukocytes andleukemia cells in the leukemic patient to determine the basis either foran impaired anti-tumor immune response or for hypercoagulability whereinthe leukemia is selected from, but not restricted to, the groupconsisting of: (a) B cell acute lymphoblastic leukemia (B-ALL); (b)Chronic lymphocytic leukemia (CLL); (c) T cell acute lymphoblasticleukemia (T-ALL); (d) Multiple myeloma; and (f) Acute myeloid leukemia(AML).

Further preferred is a method of using said antibody diagnostically in aflow cytometric analysis of Beferin expression by leukocytes in apatient with viral infection to determine the basis either for animpaired anti-viral immune response or for hypercoagulability whereinthe virus is selected from, but not restricted to, the group consistingof: (a) Cytomegalovirus; (b) Human herpes virus 6 (HHV 6); (c) Humanimmunodeficiency virus; (d) Hepatitis C virus; and (e) Hepatitis Dvirus.

Further preferred is a method of using said antibody diagnostically inan immunohistochemical analysis of Beferin expression by tissue todetermine the basis for hypercoagulability wherein said tissue isselected from, but not restricted to, the group consisting of: (a)Heart; (b) Liver; (c) Placenta; (d) Prostate; (e) Testis; (f) Uterus;(g) Pancreas; (h) Small intestine; (i) Colon; (j) Kidney; and (k) Lung.

Further preferred is a method of using said antibody diagnostically inan immunohistochemical analysis of Beferin expression by tumor cells todetermine the basis either for an impaired anti-tumor immune response orfor hypercoagulability wherein the tumor cell is selected from, but notrestricted to, the group consisting of: (a) Melanoma; (b) Breastcarcinoma; (c) Lung carcinoma; (d) Colon carcinoma; (e) Hodgkin'slymphoma; (f) Non-Hodgkin's lymphoma; (g) Prostatic carcinoma; (h)Pancreatic carcinoma; (i) Uterine carcinoma; (j) Ovarian carcinoma; (k)Testicular carcinoma; (j) Renal carcinoma; (m) Hepatic carcinoma; and(n) Lung non-small-cell carcinoma.

The efficiency of T lymphocyte co-stimulation, as well as coagulabilitystatus, can be regulated by cytokine. In a further embodiment, thepresent invention provides for the use of said Beferin antibody in invitro analysis of cytokine regulation of Beferin expression by normalleukocytes. Further preferred is a method of using said antibody in aflow cytometric analysis of said regulation by cytokine wherein thecytokine is selected from, but not restricted to, the group consistingof: (a) Interferon gamma; (b) Interleukin 17; (c) Interleukin 4; (d)Interleukin 10; (e) Interleukin 13; (f) Interleukin 15; (g) Interleukin1; (h) Interleukin 6; (i) Monocyte chemotactic protein 1 (MCP-1); (j)Vascular endothelial growth factor (VEGF); (k) Transforming growthfactor beta; (1) Interleukin 8; and (m) Tumor necrosis factor alpha.

In a further embodiment, the present invention provides for the use ofsaid Beferin antibody in in vitro analysis of cytokine regulation ofBeferin expression by non-leukocytic cell lines. Further preferred is amethod of using said antibody in a flow cytometric analysis of saidregulation by cytokine wherein the cytokine is selected from, but notrestricted to, the group consisting of: (a) Interferon gamma; (b)Interleukin 17; (c) Interleukin 4; (d) Interleukin 10; (e) Interleukin13; (f) Interleukin 15; (g) Interleukin 1; (h) Interleukin 6; (i)Monocyte chemotactic protein 1 (MCP-1); (j) Vascular endothelial growthfactor (VEGF); (k) Transforming growth factor beta; (1) Interleukin 8;and (m) Tumor necrosis factor alpha.

Further preferred is a method of contacting and specifically bindingsaid antibody with Beferin and thereby sterically inhibiting thenon-productive incorporation of Beferin into B7-H3 oligomers at the cellsurface. In so doing, said Beferin antibody up-regulates B7-H3-mediatedT lymphocyte co-stimulation. Further preferred is a method of contactingand specifically binding said antibody with Beferin and therebysterically interfering with the non-productive interaction of Beferinwith APP751, thereby un-regulating Blaa-mediated beta secretase cleavageof APP751 to generate PN2. As PN2 is an inhibitor of the extrinsiccoagulation pathway at the level of FVIIa-TF, this in turndown-regulates coagulability status. Preferred compositions comprise theBeferin antibody or fragments or derivatives thereof. Preferred route ofadministration is intravenous injection.

In a further embodiment of the invention, said Beferin antibody isincorporated as an adjuvant in vaccine preparations in a method toup-regulate the elicited immune response. In said method, said Beferinantibody facilitates the B7-H3-mediated T lymphocyte co-stimulationcontributing to establishment of specific immunity. Said Beferinantibody up-regulates B7-H3-mediated T lymphocyte co-stimulation bysterically inhibiting the non-productive incorporation of Beferin intoB7-H3 oligomers at the cell surface. Further preferred is a method touse said antibody in a vaccine targeting a viral infection wherein thevirus is selected from, but not restricted to, the group consisting of:(a) Human immunodeficiency virus; (b) Human herpes virus 6 (HHV 6); (c)Hepatitis C virus; (d) Hepatitis D virus; (e) Hepatitis E virus; (f)Cytomegalovirus; (g) Respiratory syncytial virus; (h) Herpes simplexvirus type I; (i) Herpes simplex virus type II; (j) Influenza virus; (k)Parvovirus; (m) Coxsachie virus; (n) Echovirus; (o) Epstein-Barr virus;(p) Dengue virus; (q) Lassa fever virus; and (r) Ebola virus.

Further preferred is a method to use said Beferin antibody in a vaccinetargeting a protozoan infection wherein the protozoa is selected from,but not restricted to, the group consisting of: (a) Entamoebahistolytica; (b) Cryptosporidium parvum; (c) Plasmodium falciparum; (d)Trypanosoma; (e) Leishmania; (f) Trichomonas vaginalis; and (g)Acanthamoeba.

Viruses can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Beferinantibody is used in a method of up-regulating the immune responseagainst an ongoing viral infection. In said method, said Beferinantibody facilitates the B7-H3-mediated T lymphocyte co-stimulationcontributing to the anti-viral immune response. Said Beferin antibodyup-regulates B7-H3-mediated T lymphocyte co-stimulation by stericallyinhibiting the non-productive incorporation of Beferin into B7-H3oligomers at the cell surface. Further preferred is a method ofup-regulating the immune response against an ongoing viral infectionwherein the virus is selected from, but not restricted to, the groupconsisting of: (a) Human immunodeficiency virus; (b) Human herpes virus6 (HHV 6); (c) Hepatitis B virus; (d) Hepatitis C virus; (e) Hepatitis Dvirus; (f) Cytomegalovirus; (g) Respiratory syncytial virus; (h)Influenza virus; (i) Herpes simplex virus type I; (j) Herpes simlexvirus type II; (k) Epstein Barr virus; (l) Varicella zoster virus; (m)Morbillivirus; (n) Parmyxovirus; (o) Papilloma virus; (p) Adenovirus;(q) Dengue virus; (r) Lassa fever virus; (s) Coxsachie virus; (t)Echovirus; and (u) Ebola virus.

Bacteria can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Beferinantibody is used in a method of up-regulating the immune responseagainst an ongoing bacterial infection. In said method, said Beferinantibody facilitates the B7-H3-mediated T lymphocyte co-stimulationcontributing to the anti-bacterial immune response. Said Beferinantibody up-regulates B7-H3-mediated T lymphocyte co-stimulation bysterically inhibiting the non-productive incorporation of Beferin intoB7-H3 oligomers at the cell surface. Further preferred is a method ofup-regulating the immune response against an ongoing bacterial infectionwherein the bacteria is selected from, but not restricted to, the groupconsisting of: (a) Mycobacterium avium complex; (b) Pneumocystiscarinii; (c) Acne vulgaris; (d) Legionella pneumophilia; (e) Yersiniapestis; (f) Ureaplasma urealyticum; (g) Chlamydia pneumoniae; (h)Helicobacter pylori; (i) Treponema pallidum; (j) Neisseria gonorrhoeae;(k) Salmonella typhimurium; (l) Vibrio cholera; (m) Clostridiumdifficile; (n) Bacillary dysentary; (o) Pencillin resistantPneumococcus; (p) Burkholderia mallei; (q) Mycobacterium leprae; (r)Mycobacterium haemophilum; (s) Mycobacteriun kansasii; (t) Haemophilusinfluenzae; and (u) Bacillus anthracis.

Protozoa can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Beferinantibody is used in a method of up-regulating the immune responseagainst an ongoing protozoan infection. In said method, said Beferinantibody facilitates the B7-H3-mediated T lymphocyte co-stimulationcontributing to the anti-protozoan immune response. Said Beferinantibody up-regulates B7-H3-mediated T lymphocyte co-stimulation bysterically inhibiting the non-productive incorporation of Beferin intoB7-H3 oligomers at the cell surface. Further preferred is a method ofup-regulating the immune response against an ongoing protozoan infectionwherein the protozoa is selected from, but not restricted to, the groupconsisting of: (a) Entamoeba histolytica; (b) Cryptosporidium parvum;(c) Giardia lamblia; (d) Toxoplasma gondii; (e) Isospora belli; (f)Encephalitozoon cuniculi; (g) Enterocytozoon bieneusi; (h) Plasmodiumfalciparum; (i) Trypanosoma; (j) Leishmania; (k) Trichomonas vaginalis;and (l) Acanthamoeba.

In a further embodiment of the invention, said Beferin antibody is usedin a method of up-regulating the immune response against an ongoingfungal infection wherein the fungus is selected from, but not restrictedto, the group consisting of: (a) Cryptococcal meningitis; (b)Histoplasma capstulatum; (c) Coccidiodes immitis; and (d) Candidaalbicans.

Tumors can suppress the immune response as a means of evading immunesurveillance. In a further embodiment of the invention, said Beferinantibody is used in a method of up-regulating the immune responseagainst a tumor. In said method, said Beferin antibody facilitates theB7-H3-mediated T lymphocyte co-stimulation contributing to theanti-tumor immune response. Said Beferin antibody up-regulatesB7-H3-mediated T lymphocyte co-stimulation by sterically inhibiting thenon-productive incorporation of Beferin into B7-H3 oligomers at the cellsurface. Further preferred is a method of up-regulating the immuneresponse against a tumor wherein the tumor is selected from, but notrestricted to, the group consisting of: (a) Melanoma; (b) Breastcarcinoma; (c) Lung carcinoma; (d) Colon carcinoma; (e) Prostaticcarcinoma; (f) Hodgkin's lymphoma; (g) Non-Hodgkin's lymphoma; (h)Pancreatic carcinoma; (i) Uterine carcinoma; (j) Ovarian carcinoma; (k)Testicular carcinoma; (l) Renal carcinoma; (m) Hepatic carcinoma; and(n) Lung non-small-cell carcinoma.

In a further embodiment of the invention, said Beferin antibody isincorporated as an adjuvant in therapeutic anti-tumor vaccines whereinthe tumor is selected from, but not restricted to, the group consistingof: (a) Melanoma; (b) Breast carcinoma; (c) Lung carcinoma; (d) Coloncarcinoma; (e) Prostatic carcinoma; (f) Pancreatic carcinoma; (g)Uterine carcinoma; (h) Ovarian carcinoma; (i) Testicular carcinoma; (j)Renal carcinoma; (k) Hepatic carcinoma; and (l) Lung non-small-cellcarcinoma.

Intracellular (macrophage) pathogens can be eliminated either throughmacrophage activation or through lysis of infected macrophages bycytolytic T lymphocytes (Chun et al., J. Exp. Med. 193:1213 (2001) whichdisclosure is hereby incorporated by reference in its entirety).Ligation of B7 family members expressed on the macrophage can lead tomacrophage activation [Hirokawa, M Immunol. Lett. 50:95-8 (1996), whichdisclosure is hereby incorporated by reference in its entirety]. In afurther embodiment of the invention, said Beferin antibody is used in amethod to eliminate intracellular pathogens by facilitating macrophageactivation or cytolytic T lymphocyte generation wherein the pathogen isselected from, but not restricted to, the group of intracellular(macrophage) pathogens consisting of: (a) Histoplasma capsulatum; (b)Mycobacterium tuberculosis; (c) Salmonella typhimurium; (d) Chlamydiatrachomatis; and (e) Pneumocystis carinii.

Tumors can engage the extrinsic coagulation pathway through TFexpression as a means of facilitating metastasis. In a furtherembodiment of the invention, said Beferin antibody is used in a methodof down-regulating said tumor engagement of the extrinsic coagulationpathway. In said method, said Beferin antibody facilitates Blaa-mediatedbeta secretase cleavage of APP751 to generate PN2, which is an inhibitorof the extrinsic coagulation pathway at the level of FVIIa-TF. SaidBeferin antibody facilitates Blaa-mediated generation of PN2 bysterically interfering with the non-productive interaction of Beferinwith APP751. Further preferred is a method of down-regulating tumorengagement of the extrinsic coagulation pathway wherein the tumor isselected from, but not restricted to, the group consisting of: (a)Melanoma; (b) Breast carcinoma; (c) Lung carcinoma; (d) Colon carcinoma;(e) Prostatic carcinoma; (f) Hodgkin's lymphoma; (g) Non-Hodgkin'slymphoma; (h) Pancreatic carcinoma; (i) Uterine carcinoma; (j) Ovariancarcinoma; (k) Testicular carcinoma; (l) Renal carcinoma; (m) Hepaticcarcinoma; and (n) Lung non-small-cell carcinoma.

In a further preferred embodiment, the present invention provides for amethod of screening test compounds for the ability to bind Beferin andeither inhibit or promote the capacity of Beferin to interfere withB7-H3 function. Further preferred is a method of screening said testcompounds for the ability to bind Beferin and either inhibit or promotethe capacity of Beferin to interfere with B7-H3-mediated T lymphocyteco-stimulation. Further preferred is a method of screening said testcompounds for the ability to bind Beferin and either inhibit or promotethe capacity of Beferin to interfere B7-H3-mediated T lymphocyteco-stimulation. Further preferred is a method of screening said testcompounds for the ability to bind Beferin and either inhibit or promoteB7-H3-mediated T lymphocyte co-stimulation when the antigen-presentingcell is transfected with B7-H3 and Beferin but not when the identicalcell is transfected with B7-H3 alone. Methods of screening said testcompounds and for characterizing their effect on B7-H3-mediated Tlymphocyte co-stimulation are well known to those skilled in the art.

Preferred formulation of said compound is that selected from, but notrestricted to, formulations compatible with the routes of deliveryselected from the group: (a) Oral; (b) Transdermal; (c) Injection; (d)Buccal; and (e) Aerosol.

Compounds found to bind Beferin and to inhibit the capacity of Beferinto interfere with B7-H3 function, thereby effectively up-regulatingB7-H3 activity, are used in methods analogous to those described abovefor Beferin antibody.

Compounds found to bind Beferin and to promote the capacity of Beferinto interfere with B7-H3-mediated T lymphocyte co-stimulation effectivelydown-regulate B7-H3 activity. Such compounds have application to chronicinflammatory autoimmune disease and to other disorders of immunedysregulation. Such compounds down-regulate B7-H3-mediated T lymphocyteco-stimulation by promoting the non-productive incorporation of Beferininto B7-H3 oligomers at the cell surface. In a further embodiment of theinvention, said compound is used in a method of contacting Beferin todown-regulate a dysregulated immune response and thereby treat theassociated immune disorder wherein said immune disorder is selectedfrom, but not restricted to, the group: (a) Rheumatoid arthritis; (b)Inflammatory bowel disease; (c) Insulin dependent diabetes mellitus(Type 1 diabetes); (d) Multiple sclerosis; (e) Systemic lupuserythematosus; (f) Psoriasis; (g) Allergic asthma; (h) Allergic rhinitis(hayfever); and (i) Graft versus host disease.

In a further preferred embodiment, the present invention provides for amethod of screening test compounds for the ability to bind Beferin andinhibit the capacity of Beferin to interfere with Blaa function. Furtherpreferred is a method of screening said test compounds for the abilityto bind Beferin and up-regulate Blaa-mediated PN2 generation throughAPP751 cleavage, thereby down-regulating engagement of the extrinsiccoagulation pathway by virtue of PN2 being an inhibitor of said pathway.Further preferred is a method of screening said test compounds for theability to bind Beferin and up-regulate Blaa-mediated PN2 generation byinterfering with the non-productive interaction of Beferin with APP751.Further preferred is a method of screening said test compounds for theability to bind Beferin and up-regulate PN2 release from anAPP751-expressing cell transfected with Beferin and Blaa but not fromthe identical cell line transfected with Blaa alone. Methods ofscreening said test compounds and for measuring the amount PN2 releasedinto the culture medium are well known to those skilled in the art.

Said compounds found to bind Beferin and to effect said down-regulationof the extrinsic coagulation pathway are used in methods in methodsanalogous to those described above for Beferin antibody.

Protein of SEQ ID NO:40 (Internal Designation Clone1000838788_(—)228-28-4-0-F7-F)

The cDNA of Clone 1000838788_(—)228-28-4-0-F7-F (SEQ ID NO:39) encodesthe Reductase Protein (RP):

MVSGRFYLSCLLLGSLGSMCILFTIYWMQYWRGGFAWNGSIYMFNWHPVLMVAGMVVFYGGASLVYRLPQSWVGPKLPWKLLHAALHLMAFVLTVVGLVAVFTFHNHGRTANLYSLHSWLGITTVFLFGCQWFLGFAVFLLPWASMWLRSLLKPIHVFFGAAILSLSIASVISGINEKLFFSLKNTTRPYHSLPSEAVFANSTGMLVVAFGLLVLYILLASSWKRPEPGILTDRQLLLQLRPGSRPFPVTYVSVTGRQPYKSW (SEQ ID NO:40). Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:40 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone1000838788_(—)228-28-4-0-F7-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:39descried throughout the present application also pertain to the nucleicacids included in Clone 1000838788_(—)228-28-4-0-F7-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:39, SEQ ID NO:40, and Clone 1000838788_(—)228-28-4-0-F7-F. Alsopreferred are polypeptide fragments having a biological activitydescribed herein and the polynucleotides encoding the fragments.

RP is a novel member of the cytochrome b561 family of transmembraneelectron transfer proteins. RP supplies reducing equivalents bycatalyzing the transfer of electrons across a membrane from a donor toan electron acceptor. This process depends on the interaction ofhistidine residues within the protein and transition metals (usuallyiron). Also required are cofactors to act as electron donors andacceptors. Examples of electron donors include but are not limited toascorbic acid, NADH, NADPH, flavins, and reducing polypeptides. Electronacceptors include but are not limited to semidehydroascorbic acid, NAD+,NADP+, oxidized flavin species, and electron-accepting polypeptidecomplexes. Therefore, RP requires membrane association, a transitionmetal cofactor, and electron donor/acceptor cofactors for activity.These “required components of RP activity” will be referred to hereafteras such.

Preferred embodiments of the invention include: (1) a compositioncomprising an RP polypeptide sequence of SEQ ID NO:40; (2) a compositioncomprising an RP polypeptide fragment having biological activity; (3) acomposition comprising a polynucleotide sequence of SEQ ID NO:39encoding an RP polypeptide; (4) a composition comprising apolynucleotide sequence encoding an RP polypeptide fragment havingbiological activity.

A method of reducing oxidized species of iron comprising the step of:contacting an RP polypeptide or polynucleotide construct comprisingpolynucleotides encoding an RP polypeptide with iron and a cell.Preferably, ferric iron is reduced to ferrous iron. Preferably, the cellis involved in iron-uptake. Further preferably, the cell is derived fromduodenal or small intestinal epithelium. Further preferably, the cell isa brush border enterocyte.

A method of reducing monooxygenases comprising the step of: contactingan RP polypeptide or polynucleotide construct comprising polynucleotidesencoding an RP polypeptide with a monooxygenase enzyme and a cell.Preferably, the monooxygenase is peptidylglycine alpha-amidiatingmonooxygenase (PAM). Also preferred is the monooxygenase dopaminebeta-hydroxylase (DBH). Preferably, the cell is an endocrine cell.Further preferably,the cell is a neuroendocrine cell.

A method of screening for molecules that bind and/or inhibit the abilityof RP polypeptides to transfer electrons comprising the steps: (1)contacting an RP polypeptide with a test molecule; (2) detecting testmolecule binding to said RP polypeptide; and (3) detecting test moleculeinhibiting of RP polypeptide biological activity. Preferably, a testmolecule is immobilized on a semi-solid matrix.

Also preferred is a test molecule immobilized on a solid matrix.Preferably, a test molecule binding to RP polypeptide is detected usingfluorescently-labelled RP antibody. Preferably, RP biological activityis detected using a common redox assay. Further preferably, RPbiological activity is detected using an MTT reduction assay. Alsofurther preferred is RP biological activity detected using an NBTreduction assay.

A method of inhibiting RP polypeptide-dependent electron transfercomprising the step in contacting an RP polypeptide with an RPpolypeptide inhibitor.

RP polypeptides are capable of transferring electrons to iron species,for example, reducing ferric(III) iron to ferrous(II) iron. Non-hemeassociated Fe(III) is highly insoluble in the body, while reduced Fe(II)is more readily absorbed. Thus, a method for reducing Fe(III) to Fe(II)is a highly desirable treatment for disorders such as hemolytic diseases(e.g., sickle cell anemia), hemoglobinopathies, low iron absorption,rheumatoid arthritis, hypoxia, anemias associated with pregnancy,end-stage renal failure, cancer chemotherapy, and AIDS (particularly insubjects who are being treated with zidovudine (AZT)), and chronicanemia. Furthermore, increased iron uptake enables rapid weight gaindesired in livestock. In a preferred embodiment of the invention, aniron-reducing effective amount of RP polypeptides or a polynucleotideconstruct comprising polynucleotides encoding said polypeptide are usedin a method to reduce oxidized species of iron. This method comprisesthe step of contacting a RP polypeptide or polynucleotide construct withrequired components of RP activity, iron, and cells. Preferred cells arethose involved in iron-uptake. Further preferred cells are those of theduodenum and small intestinal epithelium such as brush borderenterocytes [for review, see Siddiqi, S., et al. (2001) Curr. Opin.Gastroenterol. 17:110-7, which disclosure is hereby incorporated byreference in its entirety].

RP is expressed in neuroendocrine tissues where it is localized tosecretory vesicles. RP supplies reducing ability (i.e., electrons) tomonooxygenase enzymes, which play a role in biosynthesis and processingof catecholamines (e.g., dopamine and norepinephrine) and peptidehormones (e.g., neuropeptides, gonadotropins, somatotropins,thyrotropins, corticotropins, and lactotropins such as vasopressin,oxytocin, and insulin). In a preferred embodiment of the invention, areducing effective amount of RP polypeptides or polynucleotides encodingsaid polypeptides are used in a method to reduce monooxygenases, therebyincreasing the activity of these enzymes. This method comprises the stepof contacting a RP polypeptide or polynucleotide construct with requiredcomponents of RP activity, monooxygenase enzymes, and cells. Preferredmonooxygenase enzymes include but are not limited to peptidylglycinealpha-amidating monooxygenase (PAM) and dopamine beta-hydroxylase (DBH).Preferred cells are those that express endogenous monooxygenases, suchas cells of the adrenal medulla, pituitary gland, and other neural andendocrine tissues.

Delivery of RP polypeptide or a polynucleotide construct comprisingpolynucleotides encoding RP polypeptide to cells is accomplished bymethods common to the art such as transfection, electroporation, ormicroinjection. Additional methods of contacting said polynucleotideconstruct with cells include but are not limited to: lipid vesicledelivery (including micelles, viral envelope components, lipsomes, andmodified versions of these) as discussed in U.S. Pat. Nos. 6,110,490,5,019,369, and P.C.T. 9704748, which disclosures are hereby incorporatedby reference in their entireties; viral transduction (includingattenuated lentiviral and adenoviral systems) as discussed in U.S. Pat.No. 6,204,060, which disclosure is hereby incorporated by reference inits entirety; and delivery of naked polynucleotides (preferably to cellsof the gastrointestinal tract) as discussed in U.S. Pat. No. 6,225,290,which disclosure is hereby incorporated by reference in its entirety.

An example method of delivery comprises steps: i) compressing apolynucleotide construct, preferably comprising the polynucleotidesencoding RP polypeptide operably linked to an expression control element(e.g., a CMV promoter to direct constitutive expression), into a lipidvesicle derived from any of the following list: viral envelopes,liposomes, micelles, gangliosides and modified versions of these,preferably GM-1 ganglioside and phosphatidylserine, as described in U.S.Pat. Nos. 6,180,603, 6,110,490 or P.C.T. 9704748, which disclosures arehereby incorporated by reference in their entireties; ii) targeting thelipid vesicle to specific cells, for example, by embedding a targetingmoiety into the lipid envelope (e.g., growth hormone secretagogue forpituitary localization); iii) contacting the targeted vesicle withspecific cells by methods common to the art such as injection orinhalant (U.S. Pat. No. 6,110,490, P.C.T. 9704748, and U.S. Pat. No.6,180,603, which disclosures are hereby incorporated by reference intheir entireties).

In an additional example of delivery, a polynucleotide constructcomprising polynucleotides encoding the RP polypeptide operably linkedto an expression control element (e.g., a CMV promoter to directconstitutive expression or a brush border-specific promoter such as thesucrase promoter) is delivered orally (e.g., in aphysiologically-acceptable liquid, slurry, syrup, paste, powder, pill,or capsule form) to increase iron absorption by brush border enterocytesin the duodenum. Said naked polynucleotide construct may be modified tospecifically target certain cells of the intestine, for example, byadding an oligosaccharide modification specific for brush border celllectins (e.g., wheat germ agglutinin). Said naked polynucleotideconstruct may further provide for site-specific integration into thegenome of the target intestinal cell. For example, said construct can bemodified such that polynucleotides encoding RP polypeptide and anoperably linked promoter to are flanked by the position-specificintegration markers of Saccharomyces cerevisiae Ty3 (U.S. Pat. No.5,292,662, which disclosures are hereby incorporated by reference intheir entirety).

Further included in the present invention are methods of inhibiting theabove RP activities using an inhibitor of RP. Thus, a preferredembodiment of the present invention is a method of inhibiting RPpolypeptide-dependent electron transfer (including reduction of ferriciron to ferrous iron and reduction of monooxygenase enzymes) bycontacting RP polypeptides with RP polypeptide inhibitors. A furtherembodiment of the invention is a method of screening for compounds thatbind and/or inhibit the ability of RP polypeptides to transferelectrons. This method comprises the steps of: i) contacting an RPpolypeptide with a test compound; and ii) detecting whether said testcompound binds and/or inhibits RP polypeptide reducing activity.Detection of RP polypeptide binding is accomplished by methods common tothe art (e.g., by immobilizating said test compound on a solid orsemi-solid matrix and detecting RP polypeptides byfluorescently-labelled RP antibody). Inhibition of RP polypeptidereducing activity is measured using common assays to detect redox andelectron transfer activity, such as MTT reduction (Chakrabarti, R., etal. (2000) J. Cell Biochem. 18:133-8, which disclosure is herebyincorporated by reference in its entirety) or NBT reduction [Meerhof, L.and Roos, D. (1986) J. Leukoc. Biol. 39:699-711, which disclosure ishereby incorporated by reference in its entirety].

Protein of SEQ ID NO:42 (Internal Designation Clone1000943975_(—)160-213-2-0-A5-F)

The cDNA of Clone 1000943975_(—)160-213-2-0-A5-F (SEQ ID NO:41) encodesthe Small Secreted Serine Protease Inhibitor (SSSPI) comprising theamino acid sequence:

MPACRLGPLAAALLLSLLLFGFTLVSGTGAEKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSLPNDKEGSCPQVNINFPQLGLCRDQCQVDSQCPGQMKCCRNGCGKVSC VTPNF (SEQID) NO:42). Accordingly, it will be appreciated that all characteristicsand uses of the polypeptides of SEQ ID NO:42 described throughout thepresent application also pertain to the polypeptides encoded by thenucleic acids included in Clone 1000943975_(—)160-213-2-0-A5-F. Inaddition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:41 described throughout the presentapplication also pertain to the nucleic acids included in Clone1000943975_(—)160-213-2-0-A5-F. A preferred embodiment of the inventionis directed toward the compositions of SEQ ID NO:41, SEQ ID NO:42, andClone 1000943975_(—)160-213-2-0-A5-F. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding said fragments.

The Small Secreted Serine Protease Inhibitor (SSSPI) includes two WAP(whey acidic protein)/four-disulfide core domains, which are commonlyfound in serine protease inhibitors. SSSPI is extremely stable due tothe presence of extensive intramolecular disulfide bonds. The biologicalactivity of SSSPI is to inhibit protein degradation by serine proteasesdetermined, for instance, by tracking protein degradation by methodscommon to the art (e.g., Coomassie Blue stain). Furthermore, SSSPIactivity is associated with retarding growth in tissues that includesmooth muscle, colon, ovarian, and mammary tissues.

In a preferred embodiment of the invention, SSSPI polypeptides orfragments thereof are used to screen libraries of compounds forformation of binding complexes between SSSPI polypeptide and the agentbeing tested. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes is measuredby methods known in the art (e.g., fluorescent labeling or greenfluorescent protein tagging of the test agent, SSSPI polypeptides, orantibodies against either). A preferred method for screening providesfor high throughput screening of compounds having suitable bindingaffinity to SSSPI polypeptide. An example of this method comprises thesteps: i) synthesizing large numbers of different small test compoundsonto a solid substrate, such as plastic pins; ii) reacting testcompounds with SSSPI polypeptides and washed; iii) detecting bound SSSPIpolypeptides by methods known in the art. Alternatively, SSSPIpolypeptides are coated directly onto plates or immobilized usingnon-neutralizing antibodies and used in the aforementioned screeningtechniques. This method is applied, for example, to detecting proteaselevels in a test solution or to screening for molecules that interactwith SSSPI as discussed in the following embodiment. In anotherembodiment of the invention, binding complexes of SSSPI polypeptide andthe aforementioned test agents are used in a method to screen forcompounds that inhibit interaction of SSSPI polypeptide with serineprotease substrates. This method comprises the steps: i) allowing SSSPIpolypeptide-test agent binding complex to form; ii) adding SSSPIsubstrate (such as elastase); iii) measuring SSSPI binding to substratedirectly or indirectly by methods common in the art (e.g., fluorescentlabeling of the substrate molecule or of an antibody against saidsubstrate). This method is applied, for example, to screening formolecules that inhibit SSSPI biological activity.

In a preferred embodiment of the invention, a method of inhibitingprotein degradation with a biologically active SSSPI polypeptide or apolynucleotide construct comprising polynucleotides encoding saidpolypeptide is provided. This method comprises the step of contacting aprotein degradation-inhibiting effective amount of SSSPI polypeptidewith proteins in a solution of appropriate pH and salt concentration toallow SSSPI biological activity (e.g., buffered saline). In anadditional embodiment, SSSPI polypeptide is combined with other proteaseinhibitors and used in a method to inhibit protein degradation. Thismethod comprises the steps: combining a protein degradation-inhibitingeffective amount of SSSPI polypeptide with effective amounts of otherprotease inhibitors to form a protease inhibitor cocktail and contactingsaid cocktail with proteins in a solution of appropriate pH and saltconcentration to allow SSSPI biological activity. Preferred proteaseinhibitors are of a different specificity than SSSPI to maximize theprotease-inhibiting effectiveness of the cocktail, such as Kunitz-,trypsin inhibitor-like cystine-rich domain (TIL)-, thyroglobulin-,Kazal-, and netrin (NTR)-type protease inhibitors.

Biologically acceptable salts of the SSSPI polypeptide also fall withinthe scope of the invention. The term “biologically acceptable salts” asused herein means an inorganic acid addition salt such as hydrochloride,sulfate, and phosphate, or an organic acid addition salt such asacetate, maleate, fumarate, tartrate, and citrate. Examples ofbiologically acceptable metal salts are alkali metal salts such assodium salt and potassium salt, alkaline earth metal salts such asmagnesium salt and calcium salt, aluminum salt, and zinc salt. Examplesof biologically acceptable organic a mine addition salts are salts withmorpholine and piperidine. Examples of biologically acceptable aminoacid addition salts are salts with lysine, glycine, and phenylalanine.

Compounds provided herein can be formulated into “physiologicallyacceptable compositions” by admixture with physiologically acceptablenontoxic excipients and carriers. Such compositions may be prepared foruse in parenteral administration, particularly in the form of liquidsolutions or suspensions; oral administration, particularly in the formof tablets or capsules; intranasally, particularly in the form ofpowders, nasal drops, or aerosols; dermally, via, for example,transdermal patches; or prepared in other suitable fashions for theseand other forms of administration as will be apparent to those skilledin the art.

Common excipients include, for example, sterile water or saline,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, and hydrogenated naphthalenes. Further excipient formulationsinclude but are not limited to lactose, polyoxyethylene-9-lauryl ether,glycocholate, deoxycholate, salicylate, citric acid, oily or gel-likesolutions and lipophilic emulsions. Potentially useful parenteraldelivery systems for these active compounds include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. The invention can be employed as the sole activeagent or can be used in combination with other active ingredients whichcould facilitate inhibition of serine proteases.

Protease activity is associated with tumor formation by mechanisms thatinclude proteolytic processing of growth factors (e.g., insulin-likegrowth factor, fibroblast growth factor (FGF), epidermal growth factor(EGF), heparin-binding epidermal growth factor-like growth factor, tumornecrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta).Indeed, SSSPI is capable of inhibiting proliferation of prostatecarcinoma cells and pulmonary artery smooth muscle by preventingproteolytic processing of insulin-like growth factor II and FGF,respectively. In a preferred embodiment of the invention, a proteindegradation-inhibiting effective amount of SSSPI polypeptide iscontacted with cells to inhibit proteolytic processing and degradationof proteins. Preferred cells are those expressing growth factors thatrequire proteolytic processing to promote proliferation, such as thoselisted above. Examples of preferred cells include those from the lung,gastrointestinal tract, liver, skin, mammary gland, pancreas, ovary,prostate gland, and vascular smooth muscle and epithelia. This methodcomprises the step of contacting a physiologically acceptablecomposition of SSSPI polypeptide with cells. Delivery of saidcomposition to cells is accomplished as discussed above, as determinedappropriate by one skilled in the art.

An additional embodiment of the invention provides a method ofintroducing a polynucleotide construct comprising polynucleotidesencoding SSSPI polypeptides to cells to inhibit proteolytic processingand degradation of proteins. Preferred cells are those expressing growthfactors that require proteolytic processing to promote proliferation(e.g., insulin-like growth factor, FGF, EGF, heparin-binding epidermalgrowth factor-like growth factor, TNF-alpha, and TGF-beta) or cells thatcontact said cells. Examples of preferred cells include those from thelung, gastrointestinal tract, liver, skin, mammary gland, pancreas,ovary, prostate gland, and vascular smooth muscle and epithelia.Preferred polynucleotide constructs comprise polynucleotides encodingSSSPI polypeptide operably linked to an expression control element suchas a promoter. Preferred expression control elements direct expressionof SSSPI polypeptide in amount effective to inhibit protein degradation.Examples include the CMV promoter for constitutive expression or atissue-specific promoter, such as the human glandular kallikrein-2promoter for expression in androgen receptor-positive prostate cancercells. A physiologically acceptable composition comprising thepolynucleotide construct is introduced to cells using methods common tothe art such as electroporation or transfection. Additional deliverymethods of said physiologically acceptable composition include but arenot limited to: lipid vesicle delivery (including micelles, viralenvelope components, lipsomes, and modified versions of these) asdiscussed in U.S. Pat. Nos. 6,110,490, 5,019,369, and P.C.T. 9704748,which disclosures are hereby incorporated by reference in theirentireties; viral transduction (including attenuated lentiviral andadenoviral systems) as discussed in U.S. Pat. No. 6,204,060, whichdisclosure is hereby incorporated by reference in its entirety; anddelivery of a physiologically acceptable composition comprising nakedpolynucleotides (for example, to cells of the gastrointestinal tract) asdiscussed in U.S. Pat. No. 6,225,290, which disclosure is herebyincorporated by reference in its entirety.

SSSPI is capable of inhibiting serine proteases implicated indegenerative disorders including but not limited to thrombin, humanleukocyte elastase, pancreatic elastase, trypsin, chymase, and cathepsinG. Thrombin is produced in the blood coagulation cascade and isimplicated disorders such as thrombophlebitis, thrombosis, otherbleeding disorders, and asthma. Human leukocyte elastase is implicatedin tissue degenerative disorders such as rheumatoid arthritis,osteoarthritis, atherosclerosis, bronchitis, cystic fibrosis, andemphysema. Pancreatic elastase and trypsin are implicated soft tissuedegradation, particularly in cases of pancreatitis. Chymase, an enzymeimportant in angiotensin synthesis, is implicated in disorders such ashypertension, myocardial infarction, and coronary heart disease.Cathepsin G is implicated in abnormal connective tissue degradation,particularly in the lung. In the extreme, serine proteases including butnot limited to those mentioned above, kallikrein, and prostate specificantigen (PSA) are involved in tumor formation through proteolyticremodeling of extracellular matrix (ECM) proteins. This proteolyticremodeling may result in disruption of the integrity of tissueepithelial lining and basement membranes and result in metastasis. In apreferred embodiment of the invention, a protein degradation-inhibitingeffective amount of SSSPI polypeptides are applied to cells to inhibitprotein degradation and resulting tissue or ECM degeneration. Thismethod comprises the step of contacting a physiologically acceptablecomposition comprising SSSPI polypeptides with cells. Preferred cellsinclude those diagnosed or at risk of degenerative disorders as a resultof serine protease activity, such as those lung, gastrointestinal tract,liver, skin, mammary gland, pancreas, ovary, prostate gland, bone andcartilage, and vascular smooth muscle and epithelia. Further preferredcells include those diagnosed or at risk of tumor invasion as a resultof serine protease activity such as those involved in formation ofepithelial linings, basement membranes, and ECM (e.g., epithelial cellsand fibroblasts). Delivery of said composition to cells is accomplishedas discussed above, as determined appropriate by one skilled in the art.

In a further embodiment of the invention, SSSPI polypeptides orfragments thereof are used in a method to detect serine proteases. Thismethod is directed toward diagnosis of the aforementioned disorders anddiseases. An example of this method comprises the steps of contactingSSSPI polypeptides with a biological fluids (e.g., cell culture media,blood, serum, cell suspensions or samples) suspected of containingserine proteases, washing, and detecting serine protease-SSSPIcomplexes. Detection of said complexes is accomplished by methods commonto the art such as competition with a fluorescently-labeled neutralizingantibody.

Protein of SEQ ID NO:44 (Internal Designation Clone147441_(—)106-025-2-0-C11-F)

The cDNA of Clone 147441_(—)106-025-2-0-C11-F (SEQ ID NO:43) encodes theCarboxyPeptidase Inhibitor-1 (CPI-1):

MQGTPGGGTRPGPSPVDRRTLLVFSFILAAALGQMNFTGDQVLRVLAKDEKQLSLLGDLEGLKPQKVDFWRGPARPSLPVDMRVPFSELKD (SEQ ID NO:44). Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:44 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone147441_(—)106-025-2-0-C11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:43described throughout the present application also pertain to the nucleicacids included in Clone 147441_(—)106-025-2-0-C11-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:43, SEQ ID NO:44, and Clone 147441_(—)106-025-2-0-C11-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

CPI-1 is a 91 amino acid protein that is highly homologous to theamino-terminal “prepro” region of preprocarboxypeptidase. The “pre”region represents a signal peptide while the “pro” region inhibitscarboxypeptidase enzyme activity by binding to the active site of theenzyme before being proteolytically removed. Proteolytic cleavage ofprocarboxypeptidase results in formation of mature, activecarboxypeptidase. Proteolytic processing of procarboxypeptidase (e.g.,by trypsin) relies on the carboxy-terminus of the “pro” region, which isabsent in CPI-1. CPI-1 therefore acts as a small, independent inhibitorof carboxypeptidase activity that is not recognized bycarboxypeptidase-specific proteases. Carboxypeptidases comprise a familyof proteins that function in many physiological processes. Theseproteins remove a wide range of carboxyl-terminal amino acids, and indoing so are able to activate, inactivate, and modulate enzyme andpeptide hormone activity, as well as participate in peptide degradationand amino acid absorption. Active forms of mammalian carboxypeptidasesmay be secreted or located in lysosomes where they regulateintracellular protein processing, degradation and turnover. The“biological activity” of CPI-1 polypeptide is defined as the ability toinhibit carboxypeptidase activity. Carboxypeptidase activity may bemeasured by methods common to the art, such as incubation of a testsample with a radiolabeled Bolton-Hunter reagent-coupled peptidesubstrate (Normant, E., et al. (1995) Proc. Natl. Acad. Sci.92:12225-9). “Carboxypeptidase” is used herein to refer to any member ofthe carboxypeptidase family.

Preferred embodiments of the present invention include: (1) acomposition, comprising a CPI-1 polypeptide sequence of SEQ ID NO:44;(2) a composition, comprising a CPI-1 polypeptide fragment having acarboxypeptidase-inhibiting biological activity; (3) a composition,comprising a polynucleotide sequence of SEQ ID NO:43 encoding a CPI-1polypeptide; (4) a composition, comprising a polynucleotide sequenceencoding a carboxypeptidase-inhibiting biologically active CPI-1polypeptide fragment.

A method of inhibiting carboxypeptidase-mediated anti-fibrinolyticactivity, comprising the step of: contacting an effective amount of aCPI-1 polypeptide or biologically active fragment thereof withcarboxypeptidase in the bloodstream of an individual. Furtherpreferably, CPI-1 polypeptide is delivered to a human.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated pancreatitis, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a pancreatic cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated pancreatic cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a pancreatic cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated lung cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a lung cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated ovarian cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith an ovarian cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated larynx cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a larynx cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated uterine cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a uterine cell.

A method of preventing or inhibiting the progression ofcarboxypeptidase-mediated hepatic cancer, comprising the step of:contacting a CPI-1 polypeptide or biologically active fragment thereofwith a hepatic cell.

A method of binding an antibody or antibody fragment to a CPI-1polypeptide comprising the step of: contacting said antibody or antibodyfragment with a biological sample.

A method of using an antibody or antibody fragment that specificallybinds CPI-1 polypeptides or fragments thereof in a detection assaycomprising the steps of: contacting said antibody or antibody fragmentwith a biological sample; and detecting antibody or antibody fragmentbinding to said sample.

A further preferred method comprises the additional step of: contactinga second antibody, or antibody fragment, that does not bind CPI-1polypeptides or fragments thereof with said biological sample.

Further preferably, the first and/or second antibodies or antibodyfragments are modified with detectable molecular tags.

Further preferably, the biological sample is a blood sample or a tissuesample.

Further preferably, the detection assay is used for purposes ofdiagnosis.

A method of using an antibody or antibody fragment that binds CPI-1polypeptides or fragments thereof to inhibit CPI-1 biological activityand facilitate carboxypeptidase activity, comprising the step of:contacting said antibody or antibody fragment with CPI-1 polypeptides orbiologically active fragments thereof.

The coagulation and fibrinolytic pathways are balanced to produce bloodclotting and clot degradation, respectively, at appropriate times.Carboxypeptidase activity is anti-fibrinolytic, i.e., carboxypeptidaseabrogates clot degradation, most likely by inhibiting plasminogenactivation. In a preferred embodiment of the invention, acarboxypeptidase-inhibiting effective amount of a CPI-1 polypeptide,fragment thereof, or a polynucleotide encoding said polypeptide is usedto inhibit carboxypeptidase-mediated blood clot formation and retention.This method may be directed toward facilitating anti-coagulant activityas desired in cases such as immobilization, thrombophilia, hereditarythrombophilia, stroke, myocardial infarction, coronary artery disease,malignant conditions, during and after surgical procedures, and in casesof increased risk of blood clots associated with medications.Preferably, this method is directed toward treatment of these conditionsin a human. This method comprises the step of contacting a CPI-1polypeptide or a biologically active fragment thereof withcarboxypeptidase by administering a CPI-1 polypeptide to and individual.A preferred method of delivering CPI-1 polypeptides or biologicallyactive fragments thereof to an individual includes direct, intravenousinjection of said polypeptides or fragments in a physiologicallyacceptable solution (e.g., pH-buffered isotonic saline solutions,pH-buffered isotonic saline solutions modified by addition of viscouselements such as glycerol).

An additional preferred method of delivering CPI-1 polypeptides orfragments to an individual comprises the step of introducing apolynucleotide construct comprising polynucleotides encoding CPI-1polypeptides or biologically active fragments thereof into a cell.Preferred cells are those lining the bloodstream, such as vascularendothelial cells, vascular smooth muscle cells, and fibroblasts.Additional preferred cells are those that travel through thebloodstream, such as hematopoetic cells and their precursors,lymphocytes, macrophages, eosinophils, neutrophils, and red blood cells.Preferred polynucleotide constructs comprise an expression controlelement operably linked to polynucleotides encoding a CPI-1 polypeptideor biologically active fragment thereof. Examples of commerciallyavailable expression control units include but are not limited to a CMVpromoter for constitutive expression or a tetracycline-repressiblepromoter for regulated expression. Said polynucleotide construct isdelivered to the cell by methods determined appropriate for the celltype. Delivery to cells that travel through the bloodstream may beaccomplished by methods common to the art such as transfection orelectroporation. Cells carrying the polynucleotide construct are thenintroduced to the bloodstream by, for instance, injection. Delivery tocells that line the bloodstream may be accomplished by methods includingbut not limited to lipid vesicles or viral transduction, as described inany one of the list: U.S. Pat. Nos. 5,616,565, 6,110,490, 6,204,060, andP.C.T. 9704748 which disclosures are hereby incorporated by reference intheir entireties. Lipid vesicles may be derived from elements includingbut not limited to: viral envelopes, liposomes, micelles, and modifiedversions of these, as described in U.S. Pat. No. 6,110,490 or P.C.T.9704748, which disclosures are hereby incorporated by reference in theirentireties. Lipid vesicles or viruses may further be targeted tospecific cells, for example, by embedding a member of areceptor-receptor ligand pair into the lipid envelope (e.g., VEGF/VEGFRfor targeting to vascular endothelial cells).

While carboxypeptidase activity is required for normal proteinprocessing in the pancreas, higher than normal levels of activity leadto pancreatitis, or destruction and inflammation of the pancreas.Pancreatitis often leads to pancreatic cancer. Carboxypeptidase isactive in the extracellular space of the pancreas as well as in vacuolarcompartments such as lysosomes. In a preferred embodiment of theinvention, a carboxypeptidase-inhibiting effective amount of CPI-1polypeptides, biologically active fragments thereof, or polynucleotidesencoding said polypeptides are used to prevent or inhibit progression ofpancreatitis or pancreatic cancer. This method comprises the step ofcontacting a physiologically acceptable solution comprising a CPI-1polypeptide or biologically active fragment thereof with a pancreaticcell. Said polypeptides may be delivered, for example, by implanting aCPI-1 polypeptide—releasing stent surgically or via catheter (U.S. Pat.Nos. 5,500,013, 5,449,382, which disclosures are hereby incorporated byreference in their entireties). Polypeptides may further be delivered bydirect injection (catheter or syringe) into the pancreatic organ. Afurther preferred method of delivering CPI-1 polypeptides orbiologically active fragments thereof includes introducing apolynucleotide construct comprising polynucleotides encoding saidpolypeptides into a pancreatic cell. This method has the advantage ofcontacting CPI-1 polypeptides with intracellular compartments ofcarboxypeptidase activity. Said polynucleotide construct may furtherinclude an expression control element operably linked to polynucleotidesencoding CPI-1 polypeptides or biologically active fragments thereof.Said polynucleotide construct may be delivered to a pancreatic cell bymethods including but not limited to lipid vesicles or viraltransduction, as described in any one of the list: U.S. Pat. Nos.5,616,565, 6,110,490, 6,204,060, and P.C.T. 9704748 which disclosuresare hereby incorporated by reference in their entireties. Lipid vesiclesmay be derived from elements including but not limited to the followinglist: viral envelopes, liposomes, micelles, and modified versions ofthese, as described in U.S. Pat. No. 6,110,490 or P.C.T. 9704748, whichdisclosures are hereby incorporated by reference in their entireties.Lipid vesicles or viruses may further be targeted to specific cells, forexample, by embedding a member of a receptor-receptor ligand pair intothe lipid envelope.

Aside from pancreatic cancer, higher than normal levels ofcarboxypeptidase activity are found in cancers that include: lung,ovary, larynx, uterus, liver, stomach, and breast cancers.Carboxypeptidase activity leads to an increase in inflammatorycytokines, such as Tumor Necrosis Factor (TNF)-alpha. Therefore,carboxypeptidase-mediated tumorigenesis results from inflammation anddestruction in a number of tissue types. As a preferred embodiment ofthe invention, a carboxypeptidase-inhibiting effective amount of CPI-1polypeptides, biologically active fragments thereof, or polynucleotidesencoding said polypeptides are used to prevent or inhibit progression ofcancers. Preferred cancers include those listed above. This methodcomprises the step of contacting a physiologically acceptable solutioncomprising a CPI-1 polypeptide or biologically active fragment thereofwith a cell. Preferred cells include those of the lung, ovary, larynx,uterus, liver, stomach, and breast. Further preferred cells are those atrisk of or displaying cancerous or precancerous pathology as is commonlydetermined by those skilled in the art (e.g., loss of contactinhibition, abnormal cell size or shape). CPI-1 polypeptides,biologically active fragments thereof, or polynucleotides encoding saidpolypeptides are delivered to a specific cell by methods common to theart such as those discussed herein.

In an additional embodiment of the invention, CPI-1 polypeptides orfragments thereof are used to generate antibodies (or antibodyfragments) that specifically bind to CPI-1 polypeptides or fragmentsthereof (detAbs for “detection antibodies”) and/or inhibit thebiological activity of CPI-1 polypeptides or fragments thereof (inhAbsfor “inhibitory antibodies”). Antibodies may be polyclonal or monoclonaland may be generated by any method known to one skilled in the art.

In a preferred embodiment of the invention, antibodies or antibodyfragments that specifically bind and inhibit CPI-1 biological activity(inhAbs) are used to facilitate carboxypeptidase activity. This methodmay be directed toward increasing carboxypeptidase-mediatedanti-fibrinolytic activity for example, to prevent or treat bleedingdisorders. This method may alternatively be directed toward increasingcarboxypeptidase-mediated uptake of low density lipoprotein (LDL)particles by macrophages for example, to prevent or treat high bloodpressure or atherosclerosis. This method comprises the step ofcontacting inhAbs with CPI-1. A preferred method of contact includesinjection of a physiologically acceptable solution comprising inhAbs tothe bloodstream of an individual at risk of or suffering from a bleedingdisorder or high LDL levels.

In a further preferred embodiment of the invention, antibodies orantibody fragments that bind CPI-1 polypeptides or fragments thereof(detAbs) are used in assays to bind and/or detect CPI-1 polypeptides orfragments thereof. This method may be directed toward in vitro uses suchas purification of CPI-1 or carboxypeptidase polypeptides for drugdevelopment. An example of this method comprises the steps of:immobilizing a detAb on a solid or semi-solid matrix (e.g., sepharose);and exposing said immobilized detAb with a biological solutioncomprising proteins, preferably CPI-1 polypeptides or fragments thereof.This method may further be directed toward diagnosis of pancreatitis,pancreatic cancer, LDL-mediated disorders, and clotting disorders suchas hemophilia, thrombophilia, hereditary thrombophilia, stroke,myocardial infarction, coronary artery disease, malignant conditions,and blood clots. This method comprises the steps of: contacting a detAb,preferably a detectably-labeled detAb (e.g., conjugated to a fluorescenttag), with a biological sample, preferably a tissue or blood sample; anddetecting detAb binding to said sample. A further step of contacting asecond antibody or antibody fragment that does not bind CPI-1polypeptides or fragments thereof may be added to determine the specificnature of the protein detected by the first antibody or antibodyfragment. The second antibody or antibody fragment is preferably labeledwith a detectable molecular tag such as a fluorescent molecule. Furtherpreferably, a different molecular tag than that used by the firstantibody or antibody fragment is used with the second antibody orantibody fragment.

Protein of SEQ ID No:46 (Internal Designation Clone124610_(—)113-003-3-0-H5-F)

The polypeptides of SEQ ID NO:46 are encoded by the polynucleotides ofSEQ ID NO:45 of Clone 124610_(—)113-003-3-0-H5-F. It will be appreciatedthat all characteristics and uses of the polynucleotides of SEQ ID NO:45and polypeptides of SEQ ID NO:46, described throughout the presentapplication also pertain to the human cDNA of Clone124610_(—)113-003-3-0-H5-F and the polypeptides encoded thereby. Thegene of SEQ ID NO:45 is located on chromosome 17, encodes a humanretinoic acid-inducible regulator of growth arrest and differentiationand is hereby referred to as RET-A-MODULIN comprising the polypeptide

MTPSEGARAGTGRELEMLDSLLALGGLVLLRDSXXWEGXSLLKALVKKSALCGEQVHILGCEVSEEEFREGFDSDINNRLVYHDFFRDPLNWSKTEEAFPGGPLGALRAMCKRTDPVPVTIALDSLSWLLLRLPCTTLCQVLHAVSHQDSCPGDSSSVGKVSVLGLLHEELHGPGPVGALSSLAQTEVTLGGTMGQASAHILCRRPRQRPTDQTQWFSILPDFSLDLQEGPSVESQPYSDPHIPPVSKNAKARTRKCSLVSGHGRENKSCRGWGWGQGF. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:45, SEQID NO:46, and Clone 124610_(—)113-003-3-0-H5-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

A preferred embodiment of the invention is directed towards usingcompositions comprising RET-A-MODULIN and other preferred compositionsin a method for inhibiting neoplastic cell growth, killing neoplasticcells and treating cancer. More particularly, the invention concernsmethods and compositions to inhibit cellular proliferation of neoplasticcells, induce cytotoxicity in neoplastic cells and kill neoplastic cells(e.g., carcinomas, melanoma, and lymphoid tumors such as acutemyelocytic leukemia (AML)), wherein said methods comprises contactingcells with a proliferation-inhibiting amount of RET-A-MODULIN or othersequences of the invention. The method of suppressing neoplastic cellgrowth comprises the effects selected from the group consisting of: (a)inhibiting cell growth or proliferation; (b) killing said neoplasticcells; (c) inducing apoptosis in said neoplastic cells; (d) inducingnecrosis in said neoplastic cells; (e) preventing or inhibitingneoplastic cell invasion; and (f) preventing or inhibitingneoplasticcell metastasis. In a preferred embodiment, the neoplastic arecancerous or from a tumor. In another aspect of the invention, saidneoplastic cells is selected from the group consisting of bladdercarcinoma, hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma,ovarian carcinoma, cervical carcinoma, lung carcinoma, breast carcinoma,squamous cell carcinoma in head and neck, esophageal carcinoma, thyroidcarcinoma, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma,myeloma, sarcoma and neuroepithelioma. In yet another aspect of theinvention, said neoplastic cells are malignant or benign. Furtherincluded in the invention are the following protein sequences:

MLDSLLALGGLVLLRDSVEWEGRSLLKALVKKSALCGEQVHILGCEVSEEEFREGFDSDINNRLVYHDFFRDPLNWSKTEEAFPGGPLGALRAMCKRTDPVPVTIALDSLSWLLLRLPCTTLCQVLHAVSHQDSCPGDSSSVGKVSVLGLLHEELHGPGPVGALSSLAQTEVTLGGTMGQASAHILCRRPRQRPTDQTQWFSILPDFSLDLQEGPSVESQPYSDPHIPPVDPTTHLTFNLHLSKKEREARDSLILPFQFSSEKQQALLRPRPGQATSHIFYEPDAYYDLDQEDPDDDLDI,MLDSLLAIGGLVLLRDSVEWEGRSLLKALIKKSALRGEQVHVLGCEVSEEEFREGFDSDVNSRLVYHDLFRDPLNWSKPGEAVPEGPLKALRSMCKRTDHGSVTIALDSLSWLLCHIPCVTLCQALHALSQQNGDPGDNSLVEQVHVLGLLHEELHGPGSMGALNTLAHTEVTLSGKVDQTSASILCRRPQQRATYQTWWFSVLPDFSLTLHEGLPLRSELHPDHHTTQVDPTAHLTFNLHLSKKEREARDSLTLPFQFSSEKQKALLHPVPSRTTGRIFYEPDAFDDVDQEDPDDDLDI,SLLKALIKKSALRGEQVHVLGCEVSEEEFREGFDSDVNSRLVYHDLFRDPLNWSKPGEAVPEGPLKALRSMCKRTDHGSVTIALDSLSWLLCHIPCVTLCQALHALSQQNGDPGDNSLVEQVRVLGLLHEELHGPGSMGALNTLAHTEVTLSGKVDQTSASILCRRPQQRATYQTWWFSVLPDFSLTLHEGLPLRSELHPDHHTTQVDPTAHLTFNLHLSKKEREARDSLTLPFQFSSEKQKALLHPVPSRTTGHIFYEPDAFDDVDPEDPDDDLDI,MLDSLLAIGGLVLLRDSVEWEGRSLLKALIKKSALRGEQVHVLGCEVSEEEFREGFDSDVNSRLVYHDLFRDPLNWSKPGEAVPEGPLKALRSMCKRTDHGSVTIALDSLSWLLCHIPCVTLCQALHALSQQNGDPGDNSLVEQVHVLGLLHEELHGPGSMGALNTLAHTEVTLSGKVDQTSASILCRRPQQRATYQTWWFSVLPDFSLTLHEGLPLRSELHPDHHTTQVDPTAHLTFNLHLSKKEREARDSLTLPFQFSSEKQKALLHPVPSRTTGRIFYEPDAFDDVDQEDPDDDLDI,andMGTPGEGLGRCSHALIRGVPESLASGEGAGAGLPALDLAKAQREHGVLGGKLRQRLGLQLLELPPEESLPLGPLLGDTAVIQGDTALITRPWSPARRPEVDGVRKALQDLGLRIVEMGDENATLDGTDVLFTGREFFVGLSKWTNHRGAEIVADTFRDFAVSTVPVSGSSHLRGLCGMGGPRTVVAGSSEAAQKAVRAMAALTDHPYASLTLPDDAASDCLFLRPGLPGATPFLLHRGGS AEAL.

These embodiments also comprise the death effector domain ofRET-A-MODULIN, and other death effector domains including peptides

LVKKSALCGEQVHIL, LVKRHRLATMPPMV, LGWLCLLLLPIPLI, LHSDSGISVDSQSL,LPAGDRLTGIPSHI, LLLPLVLRALLVDV, LQPGPQLYDVMDAV, LDCVRLLLQYDAEI,LDCVRLLLQYNAEI, LLEQNDLEPGHTEL, LLEQNDLERGHTGL, MDGPRLLLLLLLGVMDRLRLLLLLILGV, LKPENILVDNDFHI, LKPENILVDRDFHI, LLLPLVLLELLVGI,LLLSLVLLALLMGI, LLLSLVLLALLMGI, LWALLILLIPIVLI, LWLLTILVLLIPLV,LLPLPVRAQLCAHL, WTELARELDFTEEQIH, WRRLARQLKVSDTKID, WKRLARELKVSEAKMD,WHQLHGKKEAYDTLIK, WRQLAGELGYKEDLID, WEPMVLSLGLSQTDIY, WAELARELQFSVEDIN,WAELARELQFSVEDIN, WRHLAGELGYQPEHID, WRHLAGELGYQPEHID, WKNCARKLGFTQSQID,WKNCARKLGFTESQID, WKEFVRRLGLSDHEID, WKEFMRFMGLSEHEIE, WKEFVRRLGLSEHEIE,WKEFMRLLGLSEHEIE, WKEFVRTLGLREAEIE, VKEFVRKNGMEEAKID, CWYQSHGKSDAYQDLIK,WQQLATAVKLYPDQVE.

A preferred embodiments of the invention comprise physiologicallyacceptable compositions and methods of treating cancer in a patient(such as prostate cancer, skin cancer/melanoma, pancreatic carcinoma,colon cancer, melanoma, ovarian cancer, liver cancer, small cell lungcarcinoma, non-small cell lung carcinoma, cervical cancer, breastcancer, bladder cancer, brain cancer, neuroblastoma/glioblastoma,leukemia, lymphoma, head and neck cancer, kidney cancer, myeloma andovarian cancer) characterized by proliferation of neoplastic cells whichcomprises administering to the patient an amount of a polypeptide of theinvention, effective to: (a) selectively induce apoptosis and/ornecrosis in such neoplastic cells and thereby inhibit theirproliferation; (b) inhibit cell growth and proliferation of theneoplastic cells; (c) inhibit invasion of the neoplastic cells; (d)inhibit metastasis of the neoplastic cells; (e) kill neoplastic cells;(f) preferentially inhibit cell growth and proliferation of theneoplastic cells; and (g) preferentially kill neoplastic cells.RET-A-MODULIN or other proteins of the invention or fragments thereofcan be used in combination with one or more of various anticancer agentsknown as cancer chemotherapeutic agents and/or radiation therapy. Theactive ingredient compound of the invention which can produce anexcellent anticancer effect can thus markedly promote the effect of theother anticancer agent or agents used in combination, to produce asynergistic effect. Therefore, even when the partner anticancer agent oragents are used in doses much smaller than the usual doses, asatisfactory anticancer effect can be obtained, whereby the adverseeffects of the partner anticancer agent or agents can be minimized. Assuch chemotherapeutic agents included but not limited to, for example,5-fluorouracil (5-FU; Kyowa Hakko Kogyo), mitomycin C (Kyowa HakkoKogyo), futraful (FT-207; Taiho Pharmaceutical), endoxan (Shionogi &Co.) and toyomycin (Takeda Chemical Industries). In addition, theapoptosis regulating composition of the present invention may beadministered with a vitamin D derivative to further enhance itscytotoxic characteristics (U.S. Pat. No. 6,087,350). The anti-canceragents of the present invention may be combined with an anti-oestrogencompound such as tamoxifen or anti-progesterone such as onapristone(see, EP 616812) in dosages known for such molecules.

The pharmaceutically and physiologically acceptable compositionsutilized in this invention may be administered by any number of routesincluding, but not limited to, parenteral, subcutaneous, intracranial,intraorbital, intracapsular, intraspinal, intracisternal, intrapulmonary(inhaled), oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means. In addition to the active ingredients, thesepharmaceutically and physiologically acceptable compositions may containsuitable physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations, which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.Easton, Pa.). Pharmaceutically and physiologically acceptablecompositions for oral administration can be formulated usingphysiologically acceptable carriers well known in the art in dosagessuitable for oral administration. Such carriers enable thepharmaceutically and physiologically acceptable compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions, and the like, for ingestion by the patient.Pharmaceutical preparations for oral use can be obtained through acombination of active compounds with solid excipient, sulting mixture isoptionally grinding, and processing the mixture of granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. Suitable excipients are carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, or sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitaniumdioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethylcellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents, which increase thesolubility of the compounds to allow for the preparation of highly,concentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art. Thepharmaceutically and physiologically acceptable compositions of thepresent invention may be manufactured in a manner that is known in theart, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use. Afterpharmaceutically and physiologically acceptable compositions have beenprepared, they can be placed in an appropriate container and labeled fortreatment of an indicated condition. For administration ofRET-A-MODULIN, such labeling would include amount, frequency, and methodof administration. Pharmaceutically and physiologically acceptablecompositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art. For anycompound, the therapeutically effective dose can be estimated initiallyeither in cell culture assays, e.g., of neoplastic cells, or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model may alsobe used to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Those of ordinary skillin the art are well able to extrapolate from one model (be it an invitro or an in vivo model). A therapeutically effective dose refers tothat amount of active ingredient, for example RET-A-MODULIN polypeptidesor other proteins of the invention or fragments thereof, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED50 (the dose therapeutically effectivein 50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutically and physiologically acceptable compositions, whichexhibit large therapeutic indices, are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration. The practitioner, in light of factorsrelated to the subject that requires treatment, will determine the exactdosage. Dosage and administration are adjusted to provide sufficientlevels of the active moiety or to maintain the desired effect. Factors,which may be taken into account, include the severity of the diseasestate, general health of the subject, age, weight, and gender of thesubject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutically and physiologically acceptablecompositions maybe administered every 3 to 4 days, every week, or onceevery two weeks depending on half-life and clearance rate of theparticular formulation. Normal dosage amounts may vary from 0.1 to100,000 micrograms, up to a total dose of about 1 g, depending upon theroute of administration. Guidance as to particular dosages and methodsof delivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc. For the prevention ortreatment of disease, the appropriate dosage of an anti-tumor agentherein will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the agent isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the agent, and thediscretion of the attending physician. The agent is suitablyadministered to the patient at one time or over a series of treatments.Animal experiments provide reliable guidance for the determination ofeffective doses for human therapy. Interspecies scaling of effectivedoses can be performed following the principles laid down by Mordenti,J. and Chappell, W. “The use of interspecies scaling in toxicokinetics”in Toxicokinetics and New Drug Development, Yacobi et al, eds., PergamonPress, New York 1989, pp. 42-96. For example, depending on the type andseverity of the disease, about 1 g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg)of an antitumor agent is an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. A typical daily dosage mightrange from about 1 g/kg to 100 g/kg or more, depending on the factorsmentioned above. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. However, other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays. Guidance as to particular dosagesand methods of delivery is provided in the literature; see, for example,U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipatedthat different formulations will be effective for different treatmentcompounds and different disorders, that administration targeting oneorgan or tissue, for example, may necessitate delivery in a mannerdifferent from that to another organ or tissue. Therapies may bedesigned to utilize RET-A-MODULIN cytotoxic properties. In particular,therapies to enhance RET-A-MODULIN expression or administration of saidpolypeptides are useful in promoting inhibition or death of cancerouscells. Cytotoxic reagents may include, without limitation, full lengthor fragment RET-A-MODULIN polypeptides, mRNA, or any compound, whichincreases RET-A-MODULIN biological activity.

Another therapeutic approach within the invention involvesadministration of RET-A-MODULIN therapeutic compositions(polynucleotide, antibody, small molecule agonist or recombinantRET-A-MODULIN polypeptide), either directly to the site of a desiredtarget cell or tissue (for example, by injection) or to a site where thecomposition will be further directed to the target cell or tissue, orsystemically (for example, by any conventional recombinant proteinadministration technique). The dosage of RET-A-MODULIN depends on anumber of factors, including the size and health of the individualpatient, but, generally, between 0.1 mg and 100 mg inclusive isadministered per day to an adult in any physiologically acceptableformulation.

In another embodiment, RET-A-MODULIN polypeptides and nucleic acidsequences find diagnostic use in the detection or monitoring ofconditions involving aberrant levels of apoptosis. For example,decreased expression of RET-A-MODULIN may be correlated with decreasedapoptosis in humans. Accordingly, a decrease or increase in the level ofRET-A-MODULIN production may provide an indication of a deleteriouscondition. Levels of RET-A-MODULIN expression may be assayed by anystandard technique such as Northern blot analysis and RT-PCR in biopsyspecimen.

These embodiments comprise methods for detection ofRET-A-MODULIN-mediated proliferation inhibition and apoptosis includingin vitro activity tests of RET-A-MODULIN or other proteins of theinvention or fragments thereof, further cellular proliferation assays,and cellular apoptosis/necrosis assays. Specific examples of apoptosisassays are also provided in the following references. Assays forapoptosis in lymphocytes are disclosed by Notebom et al., U.S. Pat. No.5,981,502, 1999, Li et al., “Induction of apoptosis in uninfectedlymphocytes by HIV-1 Tat protein”, Science 268: 429-431, 1995; Gibelliniet al., “Tat-expressing Jurkat cells show an increased resistance todifferent apoptotic stimuli, including acute human immunodeficiencyvirus-type 1 (HIV-1) infection”, Br. J. Haematol. 89: 24-33, 1995;Martin et al, “HIV-1 infection of human CD4.sup.+T cells in vitro.Differential induction of apoptosis in these cells.” J. Immunol.152:330-342, 1994; Terai et al., “Apoptosis as a mechanism of cell deathin cultured T lymphoblasts acutely infected with HIV-1”, J. Clin Invest.87: 1710-1715, 1991; Dhein et al., “Autocrine T-cell suicide mediated byAPO-1/(Fas/CD95)”, Nature 373: 438-441, 1995; Katsikis et al., “Fasantigen stimulation induces marked apoptosis of T lymphocytes in humanimmunodeficiency virus-infected individuals”, J. Exp. Med.1815:2029-2036, 1995; Westendorp et al., “Sensitization of T cells toCD95-mediated apoptosis by HIV-1 Tat and gp120”, Nature 375:497, 1995;DeRossi et al., Virology 198:234-244, 1994. Assays for apoptosis infibroblasts are disclosed by: Vossbeck et al., “Direct transformingactivity of TGF-beta on rat fibroblasts”, Int. J. Cancer 61:92-97, 1995;Goruppi et al., “Dissection of c-myc domains involved in S phaseinduction of NIH3T3 fibroblasts”, Oncogene 9:1537-44, 1994; Fernandez etal., “Differential sensitivity of normal and Ha-ras transformed C3Hmouse embryo fibroblasts to tumor necrosis factor: induction of bcl-2,c-myc, and manganese superoxide dismutase in resistant cells”, Oncogene9:2009-2017, 1994; Harrington et al., “c-Myc-induced apoptosis infibroblasts is inhibited by specific cytokines”, EMBO J. 13:3286-3295,1994; Itoh et al., “A novel protein domain required for apoptosis.Mutational analysis of human Fas antigen”, J. Biol. Chem.268:10932-10937, 1993. In vitro cellular proliferation assays comprisecultured cells such as Jurkat, HepG2, K562, or HeLa, which are treatedwith RET-A-MODULIN or fragments thereof at concentration ranges forexample from 0.5 to 25 ug/mL, and percent decrease in cellularproliferation is measured 24, 48, and 72 hours after treatment. Cellularapoptosis is measured using an apoptosis assay kit such as VYBRANT™Apoptosis Assay Kit #3 (Molecular Probes). After harvesting and washing,cells are stained with a FITC-labeled anti-RET-A-MODULIN antibody andanalyzed by FACS according to manufacturer's instructions. Cells will bestained with P1 or DAP1 to detect apoptotic nuclei. DNA fragmentationanalysis will be performed by cellular DNA extraction and Southern blotanalysis using about 1 ug of DNA and hybridized with randomly primed³²P-labeled chromosomal DNA from said cells, which had not been treated,with RET-A-MODULIN.

These embodiments also comprise the production of RET-A-MODULIN or otherproteins of the invention or fragments thereof by subcloning of saidnucleotides into an expression vector such as pCMV-neo for transfectionassays, Western blot analysis to measure protein expression, anddetection of RET-A-MODULIN-induced apoptosis by indirectimmunofluorescence and DNA fragmentation analysis. Also included in theinvention is the generation of specific antibodies against RET-A-MODULINor other proteins of the invention or fragments thereof according tomethods described in the art, wherein said antibodies can be polyclonalor monoclonal.

RET-A-MODULIN also shares homologies with two phosphorylatedmatrixproteins with the human cytomegalovirus, a pathogenic herpesviruscausing complications in patients with suppressed cellular immunefunctions and in prenatal infections (Ruger et al., J Virology61:446-453, 1987, Koretz et al., N. Engl. J. Med. 314:801-805, 1986,Bowden et al., N. Engl. J. Med. 314:1006-1010, 1986). A preferredembodiment comprises the use of RET-A-MODULIN and fragments thereofincluding

GPGPVGALSSLAQTEVTLG, EGPSVESQPYSD, EVSEEEFREGFDSDINN,TTLCQVLHAVSHQDSCPGDSSSVGKVSVLGLLHEELHGPGPVGALS, GPSVESQPYSD, CQVLHAVSH,GKVSVLGLLHEELHGPGPV for vaccination against Herpesvirus infections, aswell as a vaccine preparation against Herpesviruses such as humancytomegalovirus (HCMV) and Kaposi Sarcoma-Associated Herpesvirus/HumanHerpesvirus 8, which preparation comprises a RET-A-MODULIN protein orprotein part according to the invention and optionally one or morecarriers and adjuvants suitable for subunit vaccines. The use of aRET-A-MODULIN protein or protein part as defined above in a process forproducing RET-A-MODULIN-specific polyclonal or monoclonal antibodiesalso falls within the scope of the invention. Vaccination andimmunization generally refer to the introduction of a non-virulent agentagainst which an individual's immune system can initiate an immuneresponse, which will then be available to defend against challenge by apathogen. The immune system identifies invading “foreign” compositionsand agents primarily by identifying proteins and other large moleculesthat are not normally present in the individual. The foreign proteinrepresents a target against which the immune response is made. A furtherexample is a use of RET-A-MODULIN-specific antibodies according to theinvention for passive immunization against Herpesvirus infections, aswell as an immunization preparation for passive immunization againstHerpesvirus infections, which preparation includesRET-A-MODULIN-specific antibodies according to the invention andoptionally one or more carriers and adjuvants suitable for passiveimmunization preparations.

As regards preparative applications, one example is the use ofRET-A-MODULIN-specific antibodies according to the invention in aprocess for isolating and/or purifying RET-A-MODULIN. Routes ofadministration include, but are not limited to, intramuscular,intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraocularly and oral as well as transdermally or byinhalation or suppository. Preferred routes of administration includeintramuscular, intraperitoneal, intradermal and subcutaneous injectionas described by Pachuk et al., U.S. Pat. No. 6,235,888 (2001); see alsoNoteborn et al., U.S. Pat. No. 6,238,669 (2001), Patel et al.,Diagnostic Molecular Pathology 10:95-99 (2001), and Aoki and Tosato,Leuk Lymphoma, 41:229-237 (2001), which references are herebyincorporated in their entirety.

Proteins of SEQ ID NO:48 (Internal Designation Clone1000855165_(—)205-99-1-0-A5-F) and SEQ ID NO:52 (Internal DesignationClone 500721700_(—)204-43-4-0-H10-F)

The cDNA of clone 1000855165_(—)205-99-1-0-A5-F (SEQ ID:47) encodes theprotein of SEQ ID NO:48 comprising the amino acid sequence:

MIYTMKKVHALWASVCLLLNLAPAPLNADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTREKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPNGXQVDNYGTQLNAVNNSLTPQSTKVPSLFEFHGPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKGFIQRISKGGLIKTKRKRKKQRVKIAYEEIFVKNM. Accordingly, it willbe appreciated that all characteristics and uses of polypeptides of SEQID NO:48 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone1000855165_(—)205-99-1-0-A5-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:47described throughout the present application also pertain to the nucleicacids included in Clone 1000855165_(—)205-99-1-0-A5-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:47, SEQ ID NO:48, and Clone 1000855165_(—)205-99-1-0-A5-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The cDNA of clone 500721700_(—)204-43-4-0-H10-F (SEQ ID:51) encodes theprotein of SEQ ID NO:52 comprising the amino acid sequence:

MIYTMKKVHALWASVCLLLNLAPAPLNADSEEDEEHTIITDTELPPLKLMHSFCAFKSDDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTREKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPNGXQVDNYGTQLNAVNNSLTPQSTKVPSLFEFHGPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKGFIQRISKGGLIKTKRKRKKQRVKIAYEEIFVKNM. Accordingly, it willbe appreciated that all characteristics and uses of polypeptides of SEQID NO:52 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone500721700_(—)204-434-0-H10-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:51described throughout the present application also pertain to the nucleicacids included in Clone 500721700_(—)204-434-0-H10-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:51, SEQ ID NO:52, and Clone 500721700_(—)204-43-4-0-H10-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:48 encodes Tifapinix. The protein of SEQ IDNO:52 encodes Tifapinix-A58S. Tifapinix-A58S differs from Tifapinix inhaving serine at position 58 rather than alanine (A58S) (numbered fromthe initiating methionine of Tifapinix). It will be appreciated that thespecification, composition, and embodiments directed herein to Tifapinixalso are given to be directed as well to Tifapinix-A58S. Furthermore, itwill also be appreciated that in said specification, composition, andembodiments directed to any polypeptide of Tifapinix wherein saidpolypeptide includes alanine at position 58, that said specification,composition, and embodiments given to be directed as well to thecorresponding polypeptide of Tifapinix include amino acid serine atposition 58.

Tifapinix is a novel splice variant of tissue factor pathway inhibitor(TFPI-1). Tissue factor (TF) initiates the extrinsic coagulation pathway(U.S. Pat. Nos. 5,849,875; 5,106,833; 6,103,499; 5,773,251; 5,994,125,1999, which disclosures are hereby incorporated by reference in theirentirety). TFPI-1 is also known as lipoprotein associated coagulationinhibitor (LACI), so named because of its affinity for plasmalipoprotein.

Tifapinix has novel function as described below.

TFPI-1 is a secreted trivalent Kunitz-type plasma proteinase inhibitorthat negatively regulates the initiation of coagulation by producingactivated factor X (FXa) feedback inhibition of the catalytic complex ofactivated factor VII (FVIIa) and TF. The second Kunitz domain of TFPI-1binds and inhibits FXa, whereas the first Kunitz domain is responsiblefor the inhibition of FVIIa in the TF-FVIIa complex. The linker regionbetween Kunitz domains 1 and 2 of TFPI-1 is comprised of 20 amino acids(U.S. Pat. No. 5,849,875 which disclosures is hereby incorporated byreference in its entirety): TRDNANRIIKTTLQQEKPDF. The function of thethird Kunitz domain is unknown, although there is evidence that itcontains a heparin binding site. Heparin binding site(s) have also beenmapped carboxyl-terminal to the third Kunitz domain.

TFPI-1 directly inhibits FXa and, in a FXa-dependent fashion, producesfeedback inhibition of the TF-FVIIa catalytic complex. TFPI-1 is themajor inhibitor of the protease activity of the TF-FVIIa complex. Theallosteric promotion of TF-FVIIa binding by Kunitz domain 1 on FXabinding to Kunitz domain 2 presumably is carried out at least in partthrough the linker region between Kunitz domains 1 and 2. The findingthat the Kunitz domain 2, which binds FXa, is required for inhibition ofthe TF-VIIa complex has led to the proposal that TFPI-1 inhibitsTF-FVIIa by for ming a quaternary TF-FVIIa-FXa-TFPI-1 complex. Theformation of a quaternary complex can result from either the initialbinding of TFPI-1 to FXa, with subsequent binding to the TF-VIIa complexor, alternatively, TFPI-1 could bind directly to a preformedTF-FVIIa-FXa comples. The consequence of the formation of the quaternarycomplex is that TF can no longer participate in initiating coagulation.

Aside from it role in coagulation, FXa plays a role in inflammation. FXagenerated by TF-FVIIa has been shown to lead to pro-inflammatoryactivation of vascular endothelial cells through its cleavage ofprotease-activated receptor 2 (PAR2) (Camerer, E et al., Proc. Natl.Acad. Sci. USA 97:5255-60 (2000) which disclosure is hereby incorporatedby reference in its entirety). FXa can also elicit a pro-inflammatorycellular response by cleavage of protease-activated receptor 1 (PARI)(Kravchenko, R M Blood 97:3109-16 (2001) which disclosure is herebyincorporated by reference in its entirety). HLA-DR-restricted macrophageexpression of TF in rheumatoid synovium is believed to play a role indisease pathogenesis in part through generation of FXa (Dialynas D P etal., Arthritis and Rheumatism 41:1515-6 (1998) which disclosure ishereby incorporated by reference in its entirety).

TF is a bifunctional molecule capable of inducing both fibrin depositionand angiogenesis in cancer. Cancer patients are prone to venousthromboembolism, and this hypercoagulability favors tumor growth andmetastasis. In human lung cancer, melanoma, and breast cancer, TF andvascular endothelial growth factor (VEGF) co-localize in tumor cells; aclose correlation exists between TF and VEGF synthesis in tumor celllines and with angiogenesis in vivo in a severe, combinedimmunodeficient mouse model (Rickles, F R et al., Int. J. Hematol.73:145-50 (2001); Wojtukiewicz M Z et al., Thromb. Haemost. 82:1659-62(1999); Abdulkadir S A, et al., Hum. Pathol. 31:443-7 (2000); Koomagi Ret al., Int. J. Cancer 79:19-22 (1998) which disclosures are herebyincorporated by reference in their entirety).

TF supports metastasis (Mueller B M et al., J. Clin. Invest. 101: 1372-8(1998); Fischer EG et al., J. Clin. Invest. 104:1213-21 (1999) whichdisclosures are hereby incorporated by reference in their entirety).Equally important for this process are (a) interactions of the TFcytoplasmic domain, which binds the mobility-enhancing actin-bindingprotein 280, and (b) formation of a proteolytically active TF-FVIIacomplex on the tumor cell surface. In primary bladder carcinoma cells,this complex localizes to the invasive edge, in proximity totumor-infiltrating vessels that stain intensely for TFPI-1. Tumor celladhesion and migration was shown in vitro to be supported by interactionof TF-FVIIa with TFPI-1 immobilized heparin.

TF antigen has been detected in all cellular elements comprising theatheriosclerotic plaque. The most abundant sources of TF appear to bethe macrophages and intimal smooth muscle cells located in the capsurrounding the lipid-rich necrotic core. TF antigen is also present inthe medial and endothelial cells overlying the plaque. In addition toits association with vascular cells, TF antigen is also found in theextracellular matrix of the intima and in the necrotic core. This TF maycome in contact with circulating blood when the plaque ruptures-the mostimportant precipitant of acute arterial thrombosis (Taubman M B et al.,Thrombosis and Haemostasis 82:801-5 (1999) which disclosure is herebyincorporated by reference in its entirety).

Recently it has been shown that TFPI-1 inhibits the proliferation ofbasic fibroblast growth factor-stimulated endothelial cells. A truncatedform of TFPI-1, containing only the first two Kunitz-type proteinaseinhibitor domains, has very little antiproliferative activity,suggesting that the carboxyl-terminal region of TFPI-1 is responsiblefor this activity (Hembrough, T A et al., J. Biol. Chem. 276:12241-8(2001) which disclosure is hereby incorporated by reference in itsentirely). By virtue of this activity, TFPI-1 is an inhibitor ofangiogenesis. Anomalous angiogeneisis plays an important role in anumber of pathologies, including cancer, proliferative diabeticretinopathy, and rheumatoid arthritis (Folkman, J, Forum (Geneva) 9(3Suppl 3):59-62 (1999); Danis, R P et al., Expert Opin. Pharmacother2:395-407 (2001); Stupack, D G et al., Braz J. Med. Biol. Res. 32:573-81(1999) which disclosures are hereby incorporated by reference in theirentirety).

In the case of Tifapinix, alternative splicing results in the internaldeletion of exon 5 comprised of 13 amino acids from the linker regionbetween Kunitz domains 1 and 2 (Girard, T J et al., J. Biol. Chem.266:503641 (1991) which disclosure is hereby incorporated by referencein its entirety). The A58S amino acid substitution that distinguishesTifapinix-A58S from Tifapinix, as well as from the canonical TFPI-1amino acid sequence (NCBI Accession No. P10646 which disclosure ishereby incorporated by reference in its entirety), establishes that thealternative splicing of TFPI-1 represented by Tifapinix can occur formore than one allele of TFPI-1, thereby supporting the thesis that thealternative splicing represented by Tifapinix plays a significant andunique role in TFPI-1 biology.

The resultant shortened linker region between Kunitz domains 1 and 2 iscomprised of 7 amino acids: TREKPDF. The deletion also results in thegeneration of a novel amino acid neighborhood around the two amino acidsbracketing the deletion (RE, underlined above). Tifapinix retains thecapacity to bind to FXa (Kunitz domain 2), but has lost the capacity toallosterically promote binding of Kunitz domain 1 to TF-FVIIa inresponse to the FXa binding. As Tifapinix retains the capacity toinhibit FXa, Tifapinix therefore remains both an anti-coagulant and ananti-inflammatory. As the carboxyl terminus of Tifapinix remains intact,Tifapinix retains the capacity to inhibit angiogenesis.

Importantly however and in contradistinction to TFPI-1, by virtue ofhaving lost the capacity to allosterically promote TF-FVIIa-binding byKunitz domain 1, Tifapinix has lost the capacity to be recruited byTF-FVIIa for promotion of tumor cell metastasis (Mueller B M et al., J.Clin. Invest. 101:1372-8 (1998); Fischer E G et al., J. Clin. Invest.104:1213-21 (1999) which disclosures are hereby incorporated byreference in their entirety).

In a preferred embodiment, the present invention provides for anantibody that specifically binds Tifapinix of the present invention.Further preferred is a method for making said antibody wherein saidantibody recognizes a non-conformational or conformational epitope ofTifapinix.

Further preferred is a method for making said antibody wherein a mouseis immumunized with Tifapinix. Further preferred is a method whereinmonoclonal antibodies derived from said mouse are screened for bindingto Tifapinix but not to TFPI-1. Further preferred is a method of makingsaid antibody wherein said antibody is directed to the novel linkerregion sequence of Tifapinix comprised of amino acids 105-111, numberedfrom the initiating methionine of Tifapinix, or any fragment thereof.Further preferred is a method wherein monoclonal antibodies derived fromsaid mouse are screened by sandwich enzyme-linked immunosorbent assay(ELISA) for binding to Tifapinix but not to TFPI-1. Methods ofgenerating said monoclonal antibody and of establishing its specificityby methods including sandwich ELISA are well known to those skilled inthe art.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Tifapinix.Further preferred is a method for using said antibody diagnostically todetermine the basis either for immune dysfunction or forinflammopathology. In the case of inflammopathology, of which thedisease states below are representative, the level of Tifapinixexpression is expected to be depressed. In the case of non-inflammatoryimmune dysregulation, Tifapinix status is more difficult to predict apriori. In either case, Tifapinix status is expected to facilitatediagnosis and, moreover, facilitate stratification of disease states.Furthermore, Tifapinix status may also have prognostic value. Furtherpreferred is a method of using said antibody diagnostically in asandwich ELISA format to quantitate Tifapinix in plasma or other bodilyfluid, including but not restricted to synovial fluid and cerebrospinalfluid, within a pathological context. Further preferred is a method ofusing said diagnostic assay to determine the level of Tifapinix inplasma or other bodily fluid of a patient with either dysregulatedimmune function or inflammopathology wherein the immune dysfunction orinflammopathology is selected from, but not restricted to, the groupconsisting of: (a) Rheumatoid arthritis; (b) Atheriosclerosis; (c)Inflammatory bowel disease; (d) Insulin dependent diabetes mellitus(Type 1 diabetes); (e) Systemic lupus erythematosus; (f) Multiplesclerosis; (g) Psoriasis; (h) Allergic asthma; (i) Reperfusion injury;and (j) Stroke.

In further preferred embodiment, the present invention provides for amethod of using Tifapinix to treat patients with immune dysfunction orinflammopathology. Preferred compositions comprise Tifapinix. Furtherpreferred compositions comprise Tifapinix. Preferred formulation of saidcomposition is that formulation compatible with the route of deliverywherein said route of delivery is selected from, but not restricted to,the group: (a) Oral; (b) Transdermal; (c) Injection wherein injection isselected from, but not restricted to, the group consisting of:intravenous, intramuscular, subcutaneous, intra-synovial, andintra-tumoral; (d) Buccal; and (e) Aerosol.

Neovascularization plays a role in the pathogenesis of a number ofdiseases, including but not restricted to rheumatoid arthritis [Danis RP et al., Expert Opin. Pharmacother. 2:395-407 (2001) which disclosureis hereby incorporated by reference in its entirety].

In a further embodiment of the invention, said composition comprised ofTifapinix is used in a method of treating said patients with immunedysfunction or inflammopathology. Further preferred is a method oftreating said patients in a method of ameliorating the symptoms orpathology associated with said immune dysfunction or inflammopathology.Further preferred is a method of treating said patients in a method ofameliorating the symptoms or pathology associated with pathogeneticengagement of the extrinsic coagulation pathway or the promotion ofangiogenesis by TF. Further preferred are compositions comprised ofTifapinix used in methods of delivering to said patients an ameliorativeeffective amount of Tifapinix by said route of delivery. Furtherpreferred is a method of delivering said composition comprisingTifapinix by said route of delivery to patients with immune dysfunctionor inflammopathology wherein the immune dysfunction or inflammopathologyis selected from, but not restricted to, the group: (a) Rheumatoidarthritis; (b) Atheriosclerosis; (c) Inflammatory bowel disease; (d)Insulin dependent diabetes mellitus (Type 1 diabetes); (e) Systemiclupus erythematosus; (f) Psoriasis; (g) Multiple sclerosis; (h) Allergicasthma; (i) Reperfusion injury; and (j) Stroke.

In acute myocardial infarction (AMI), the monocyte TF procoagulantactivity is increased and may contribute to the risk for recurrence andother thrombotic events [Ott I et al., Blood 97:3721-6 (2001) whichdisclosure is hereby incorporated by reference in its entirety]. In afurther embodiment of the invention, said composition comprised ofTifapinix is used in a method to treat patients with AMI. Furtherpreferred is a method of delivering by intravenous injection anameliorative effective amount of Tifapinix in a method to treat patientswith AMI.

Studies confirm the important role of TF-mediated coagulation in thesmooth muscle proliferation and neointimal thickening that followsvascular injury [Han X et al., Arterioscler. Thromb. Vasc. Biol.19:2563-7 (1999); Taubman M B et al., Thrombosis and Haemostasis82:801-5 (1999) which disclosures are hereby incorporated by referencein their entirety]. In a further embodiment of the invention, saidcomposition comprised of Tifapinix is used in a method to treat patientswith neointimal thickening following vascular injury, including but notrestricted to that consequential to balloon-induced vascular injury.Further preferred is a method of delivering by intravenous injection anameliorative effective amount of Tifapinix in a method to treat patientswith intimal thickening following vascular injury.

Studies confirm the important role of TF engagement of the extrinsiccoagulation pathway in vascular pathology. In a further embodiment ofthe invention, said composition comprised of Tifapinix is used in amethod to treat patients with said TF-associated vascular pathology.Further preferred is a method of delivering by intravenous injection anameliorative effective amount of Tifapinix in a method to treat patientswith said vascular pathology. Further preferred is a method ofdelivering by intravenous injection an ameliorative effective amount ofTifapinix in a method to treat patients with said vascular pathologywherein said pathology is selected from, but not restricted to, thegroup consisting of: (a) Disseminated intravascular coagulation (DIC);(b) Hypercoagulability; and (c) Septic shock.

Proliferative diabetic retinopathy (PDR) remains one of the major causesof aquired blindness in developed nations. The hallmark of PDR isneovascularization, abnormal angiogenesis that may ultimately causesevere vitreous cavity bleeding and/or retinal detachment. In a furtherembodiment of the invention, said composition comprised of Tifapinix isused in a method to treat patients with said PDR.

In a further embodiment of the invention, said composition comprised ofTifapinix is used in a method of anti-angiogenesis or anti-metastasis totreat patients with cancer. Further preferred is a method of treatingsaid patients in a method of ameliorating the symptoms or pathologyassociated with said cancer. Further preferred are compositionscomprised of Tifapinix used in methods of delivering to said patients anameliorative effective amount of Tifapinix by said route of delivery.Further preferred is a method of delivering Tifapinix by said route ofdelivery to patients with cancer wherein the cancer is selected from,but not restricted to, the group: (a) Melanoma; (b) Breast carcinoma;(c) Lung carcinoma; (d) Colon carcinoma; (e) Prostatic carcinoma; (f)Hodgkin's lymphoma; (g) Non-Hodgkin's lymphoma; (h) Pancreaticcarcinoma; (i) Uterine carcinoma; (j) Ovarian carcinoma; (k) Testicularcarcinoma; (l) Renal carcinoma; (m) Hepatic carcinoma; and (n) Lungnon-small-cell carcinoma.

Tifapinix represents a uniquely valuable reagent with which to addressthe molecular basis for the allosteric relationship between the initialFXa binding to Kunitz domain 2 and the subsequent TF-FVIIa binding toKunitz domain 1. That is because the lesion is Tifapinix is small andwell-defined: a deletion of 13 contiguous amino acids from the linkerregion between Kunitz domains 1 and 2. Specifically, the relativeimportance of linker length and linker amino acid composition can bereadily addressed. In further preferred embodiment, therefore, thepresent invention provides for a method of recombinant DNA manipulationof polynucleotides encoding Tifapinix to identify the critical molecularparameters for said allosteric mechanism. Methods of manipulatingnucleic acid sequence, including but not restricted to site-specificmutagenesis, and expression of recombinant protein are well known tothose skilled in the art.

The capacity of Tifapinix to specifically inhibit the serine proteaseactivity of FXa makes it a very useful reagent for assessing the roleeither of FXa serine protease activity or more generally that of theactive site of FXa in a number of activities. These activities includebut are not necessarily restricted to the group:

-   -   Amplification of extrinsic coagulation, as read out in a        clotting assay (Dialynas D P et al. Cellular Immunology        177:671-9 (1997) which disclosure is hereby incorporated by        reference in its entirety;    -   Serine proteolytic cleavage of specific substrate; and    -   Docking with its receptor, EPR-1, expressed on vascular        endothelial cells and smooth muscle cells (Nicholson A C et        al., J. Biol. Chem. 271:28407-13 (1996) which disclosure is        hereby incorporated by reference in its entirety).

Whereas (a) and (b) require the active site of FXa, (c) does not.Tifapinix therefore would be a discriminating reagent with which toassess the involvement of FXa active site in diverse activities. Forexample, Tifapinix blocks (a) and (b), but does not block (c). Infurther preferred embodiment, the present invention provides for amethod of using Tifapinix to investigate the requirement for FXa activesite, and by inference FXa, in an activity manifested by a test sample.

In further preferred embodiment, Tifapinix is used for plasmin bindingand inhibition. Any suitable method may be used to test the compounds ofthis invention (U.S. Pat. No. 6,103,499, 2000). Scatchard (Ann N.Y. AcadSci (1949) 51:660-669) described a classical method of measuring andanalyzing binding, which is applicable to protein binding. This methodrequires relatively pure protein and the ability to distinguish boundprotein from unbound. A second appropriate method of measuring K.sub.Dis to measure the inhibitory activity against the enzyme. If the K.sub.Dto be measured is in the 1 nM to 1 muM range, this method requireschromogenic or fluorogenic substrates and tens of micrograms tomilligrams of relatively pure inhibitor. For the proteins of thisinvention, having K.sub.D in the range 5 nM to 50 pM, nanograms tomicrograms of inhibitor suffice. When using this method, the competitionbetween the inhibitor and the enzyme substrate can give a measuredK.sub.i that is higher than the true K.sub.i. Measurement reported hereis not so corrected because the correction would be very small and theany correction would reduce the K.sub.i. Here, we use the measuredK.sub.i as a direct measure of KD. Tifapinix has a K.sub.D for plasminof at most about 5 nM, more preferably at most about 300 pM, and mostpreferably 100 pM or less. Preferably, the binding is inhibitory so thatK.sub.i is the same as K.sub.D. The K.sub.i of QS4 for plasmin is about2 nM. The K.sub.i of SPI11 for plasmin is about 88 pM.

In another preferred embodiment, Tifapinix is used for pharmaceuticalmethods and preparations. The preferred subject of this invention is amammal. The invention is particularly useful in the treatment of humans,but is suitable for veterinary applications, too. Herein, “protection”includes “prevention”, “suppression”, and “treatment”. “Prevention”involves administration of drug prior to the induction of disease.“Suppression” involves administration of drug prior to the clinicalappearance of disease. “Treatment” involves administration of drug afterthe appearance of disease. In human and veterinary medicine, it may notbe possible to distinguish between “preventing” and “suppressing” sincethe inductive event(s) may be unknown or latent, or the patient is notascertained until after the occurrence of the inductive event(s). We usethe term “prophylaxis” as distinct from “treatment” to encompass“preventing” and “suppressing”. Herein, “protection” includes“prophylaxis”. Protection need not by absolute to be useful. Tifapinixor fragments thereof may be administered, by any means, systemically ortopically, to protect a subject against a disease or adverse condition.For example, administration of such a composition may be by anyparenteral route, by bolus injection or by gradual perfusion.Alternatively, or concurrently, administration may be by the oral route.A suitable regimen comprises administration of an effective amount ofthe protein, administered as a single dose or as several doses over aperiod of hours, days, months, or years. The suitable dosage of aprotein of this invention may depend on the age, sex, health, and weightof the recipient, kind of concurrent treatment, if any, frequency oftreatment, and the desired effect. However, the most preferred dosagecan be tailored to the individual subject, as is understood anddeterminable by one of skill in the art, without undue experimentationby adjustment of the dose in ways known in the art. For methods ofpreclinical and clinical testing of drugs, including proteins, see,e.g., Berkow el al, eds., The Merck Manual, 15^(th) edition, Merck andCo., Rahway, N.J., 1987; Goodman et al, eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985), which referencesare hereby incorporated in their entirety. In addition to Tifapinix, apharmaceutical composition may contain pharmaceutically acceptablecarriers, excipients, or auxiliaries. See, e.g., Berker, supra, Goodman,supra, Avery, supra and Ebadi, supra.

In yet another preferred embodiment, Tifapinix or fragments thereof areused for in vitro diagnostic methods and reagents. Tifapinix and relatedsequences may be applied in vitro to any suitable sample that mightcontain plasmin to measure the plasmin present. The assay must include aSignal Producing System (SPS) providing a detectable signal that dependson the amount of plasmin present. The signal may be detected visually orinstrumentally. Possible signals include production of colored,fluorescent, or luminescent products, alteration of the characteristicsof absorption or emission of radiation by an assay component or product,and precipitation or agglutination of a component or product. Thecomponent of the SPS most intimately associated with the diagnosticreagent is called the “label”. A label may be, e.g., a radioisotope, afluorophore, an enzyme, a co-enzyme, an enzyme substrate, anelectron-dense compound, or an agglutinable particle. A radioactiveisotope can be detected by use of, for example, a gamma. counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful are .sup.3 H, .sup.125 I, .sup.131 I, .sup.35 S,.sup.14 C, and, preferably, .sup.125 I. It is also possible to label acompound with a fluorescent compound. When the fluorescent-labeledcompound is exposed to light of the proper wavelength, its presence canbe detected. Among the most commonly used fluorescent labeling compoundsare fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine. Alternatively,fluorescence-emitting metals, such as .sup.125 Eu or other anthanide,may be attached to the binding protein using such metal chelating groupsas diethylenetriaminepentaacetic acid or ethylenediamine-tetraaceticacid. The proteins also can be detectably labeled by coupling to achemiluminescent compound, such as luminol, isolumino, theromaticacridinium ester, imidazole, acridinium salt, and oxalate ester.Likewise, a bioluminescent compound, such as luciferin, luciferase andaequorin, may be used to label the binding protein. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Enzyme labels, such as horseradish peroxidase and alkalinephosphatase, are preferred. There are two basic types of assays:heterogeneous and homogeneous. In heterogeneous assays, binding of theaffinity molecule to analyte does not affect the label; thus, todetermine the amount of analyte, bound label must be separated from freelabel. In homogeneous assays, the interaction does affect the activityof the label, and analyte can be measured without separation. Tifapinix,as a plasmin-binding protein may be used diagnostically in the same waythat an antiplasmin antibody is used. Thus, depending on the assayformat, it may be used to assay plasmin, or, by competitive inhibition,other substances which bind plasmin. The sample will normally be abiological fluid, such as blood, urine, lymph, semen, milk, orcerebrospinal fluid, or a derivative thereof, or a biological tissue,e.g., a tissue section or homogenate. If the sample is a biologicalfluid or tissue, it may be taken from a human or other mammal,vertebrate or animal, or from a plant. The preferred sample is blood, ora fraction or derivative thereof. In a related embodiment, Tifapinix orfragments thereof is immobilized, and plasmin in the sample is allowedto compete with a known quantity of a labeled or specifically labelableplasmin analogue. The “plasmin analogue” is a molecule capable ofcompeting with plasmin for binding to Tifapinix or fragments thereof. Itmay be labeled already, or it may be labeled subsequently byspecifically binding the label to a moiety differentiating the plasminanalogue from plasmin. The phases are separated, and the labeled plasminanalogue in one phase is quantified. In a “sandwich assay”, both aninsolubilized plasmin-binding agent (PBA), and a labeled PBA areemployed. The plasmin analyte is captured by the insolubilized PBA andis tagged by the labeled PBA, forming a tertiary complex. The reagentsmay be added to the sample in any order. The PBAs may be the same ordifferent, and only one PBA needs to comprise Tifapinix or fragmentsthereof according to this invention (the other may be, e.g., anantibody). The amount of labeled PBA in the tertiary complex is directlyproportional to the amount of plasmin in the sample. The two embodimentsdescribed above are both heterogeneous assays. A homogeneous assayrequires only that the label be affected by the binding of Tifapinix orfragments thereof to plasmin. The plasmin analyte may act as its ownlabel if Tifapinix or fragments thereof are used as a diagnosticreagent. A label may be conjugated, directly or indirectly (e.g.,through a labeled anti-Tifapinix antibody), covalently (e.g., with SPDP)or noncovalently, to the plasmin-binding protein, to produce adiagnostic reagent. Similarly, the plasmin-binding protein may beconjugated to a solid phase support to form a solid phase (“capture”)diagnostic reagent. Suitable supports include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, and magnetite.The carrier can be soluble to some extent or insoluble for the purposesof this invention. The support material may have any structure so longas the coupled molecule is capable of binding plasmin.

In yet another preferred embodiment, Tifapinix or fragments thereof areused for in vivo diagnostic uses. Tifapinix or fragments thereof, i.e. aKunitz domain that binds very tightly to plasmin can be used for in vivoimaging. Radiolabeled Tifapinix may be administered to a human or animalsubject, typically by injection, e.g., intravenous or arterial othermeans of administration such as subcutaneous, intramuscular in aquantity sufficient to permit subsequent dynamic and/or static imagingusing suitable radio-detecting devices. The dosage is the smallestamount capable of providing a diagnostically effective image, and may bedetermined by means conventional in the art, using known radio-imagingagents as guides. Typically, the imaging is carried out on the wholebody of the subject, or on that portion of the body or organ relevant tothe condition or disease under study. The radiolabeled binding proteinhas accumulated. The amount of radiolabeled binding protein accumulatedat a given point in time in relevant target organs can then bequantified. A particularly suitable radio-detecting device is ascintillation camera, such as a. gamma. camera. The detection device inthe camera senses and records (and optional digitizes) the radioactivedecay. Digitized information can be analyzed in any suitable way, manyof which are known in the art. For example, a time-activity analysis canillustrate uptake through clearance of the radiolabeled binding proteinby the target organs with time. The radioisotope used should preferablybe pharmacologically inert, and the quantities administered should nothave substantial physiological effect. The binding protein may beradio-labeled with different isotopes of iodine, for example .sup.123 I,.sup.125 I, or .sup.131 I (see, for example, U.S. Pat. No. 4,609,725).The amount of labeling must be suitably monitored.

In applications to human subjects, it may be desirable to useradioisotopes other than .sup. 125 I for labeling to decrease the totaldosimetry exposure of the body and to optimize the detectability of thelabeled molecule. Considering ready clinical availability for use inhumans, preferred radio-labels include: .sup.99m Tc, .sup.67 Ga, .sup.68Ga .sup.90 Y, .sup.111 In, .sup.113m In, .sup.123 I, .sup.186 Re,.sup.188 Re, or .sup.211 At. Radiolabeled protein may be prepared byvarious methods. These include radio-halogenation by the chloramine-T orlactoperoxidase method and subsequent purification by high pressureliquid chromatography, for example, see Gutkowska et al in“Endocrinology and Metabolism Clinics of America 16 (1):183, 1987. Othermethods of radiolabeling can be used, such as IODOBEADS.TM. Tifapinix orfragments thereof may also be used to purify plasmin from a fluid, e.g.,blood. For this purpose, it is preferably immobilized on an insolublesupport. Such supports include those also useful in preparing solidphase diagnostic reagents. Proteins can be used as molecular weightmarkers for reference in the separation or purification of proteins.

These embodiments also relate to isolation, purification and productionof antibodies wherein antibodies can be polyclonal or monoclonal asdescribed (U.S. Pat. No. 6,171,587 B1, 2000), hereby enclosed in theirentirety.

Another preferred embodiment relates to the use of Tifapinix and Kunitzdomains thereof for the inhibition of kallikrein activity. Kallikreinsare serine proteases found in both tissues and plasma (see U.S. Pat. No.5,994,125, 1999, U.S. Pat. No. 6,057,287, 2000) which references arehereby enclosed in their entirety). Plasma kallikrein is involved incontact-activated coagulation, fibrinolysis, hypotension, andinflammation mediated through the activities of factor XII(coagulation), pro-urokinase/plasminogen (fibrinolysis), and kininogens(hypotension and inflammation). Kallikrein cleavage of kininogensresults in the production of highly potent bioactive peptides (kinins),which cause increased vascular permeability, vasodilation, bronchospasm,and pain induction. Thus, kinins mediate life-threatening vascular shockand edema associated with bacteremia (sepsis) or trauma, asthma, andinflammatory and neurogenic pain associated with tissue injury, andedema in C1-inhibitor-deficient diseases (hereditary angioedema).Tifapinix, as a protease inhibitor, and fragments thereof said Kunitzdomains, prevent the cleavage of kallikrein and thus the release of saidkinins.

Tifapinix may be used for any of the foregoing purposes. Methods forproduction using eukaryotic and prokaryotic expression systems have beenreported previously and are well known in the art (U.S. Pat. No.6,103,500, 2000; PCT WO 95/18830). For example, Tifapinix or fragmentsthereof, whereas preferred fragments comprise said Kunitz domains,preferably Kunitz domain three, may be produced by any conventionaltechnique including (i) nonbiological synthesis by sequential couplingof component amino acids, (ii) production by recombinant DNA techniquesin a suitable host cell such as bacterial, insect- or mammalian cells,(iii) removal of undesired sequences from LACI and in coupling ofsynthetic replacement sequences (U.S. Pat. No. 5,994,125, 1999, herebyincorporated in its entirety).

Protein of SEQ ID NO:50 (Internal Designation Clone588098-184-11-4-0-H4-F)

The cDNA of Clone 588098_(—)184-114-0-H4-F (SEQ ID NO:49) encodes theprotein of SEQ ID NO:50 comprising the amino acid sequence

MPSSVSWGILLLAGLCCLVPVSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIDXNTKSPLFMGKVVNPTQK. Accordingly it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:50 described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in Clone588098_(—)184-11-4-0-H4-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:49described throughout the present application also pertain to the nucleicacids included in Clone 588098_(—)184-11-4-0-H4-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:49, SEQ ID NO:50, and Clone 588098_(—)184-11-4-0-H4-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:50 encodes CrypAAT, a splice variant ofalpha-1-antitrypsin (antitrypsin) with novel function. In CrypAAT,internal splicing within exon 2 leaves the signal sequence intact butresults in an N-terminal deletion of 67 amino acids from the matureprotein. This deletion extends from the disordered N-terminus throughhelix A and into helix B (Stein, P E et al., Nature Structural Biology2:96-113 (1995) which disclosure is hereby incorporated by reference inits entirety). The Met-Ser active site near the C-terminus is intact.

Antitrypsin is synthesized primarily by hepatocytes and is the mostabundant proteinase inhibitor in human plasma. Although it diffusesthrough all organs, and inhibits a large number of proteases, itsprimary function is in the lung parenchyma, where it protects alveolartissue from damage by neutrophil elastase, a serine protease released inthe course of an inflammatory response. Elastases are defined by theirability to cleave elastin, the matrix protein that gives tissues theproperty of elasticity. If left uncontrolled, neutrophil elastase leadsto excessive inflammation and progressive emphysema. Individuals withantitrypsin deficiency have at least a 20-fold increase risk ofdeveloping emphysema.

Antitrypsin is a member of the serpin (serine protease inhibitor)supergene family. The primary function of most of the serpins is theregulation of proteolytic enzymes under both physiological andpathological conditions. On the basis of strong sequence similarities, anumber of proteins with no known inhibitory activity have beenclassified as serpins. For example, thyroxine binding globulin (TBG) andcorticosteroid binding globulin (CBG) serve as transporters oflipophilic hormones, and angiotensinogen is a peptide hormone precursor(Janciauskiene, S, Biochimica et Biophysica Acta. 1535:221-35 (2001)which disclosure is hereby incorporated by reference in its entirety).

Serpins are competitive, irreversible inhibitors of serine proteases.Serpins have a common molecular design based on a five-strandedbeta-sheet A and the reactive loop arising from it, that presents apeptide sequence to the target proteinase. The function of antitrypsinas a proteinase inhibitor depends on its undergoing conformationalchange when it binds to neutrophil elastase. This change involves theinsertion of the cleaved reactive loop as the 4^(th) strand in itsbeta-sheet A, and deactivates neutrophil elastase by swinging it fromthe top to the bottom of the antitrypsin molecule (described as amousetrap action) (Parmar, J S et al., Journal of the Royal College ofPhysicians of London 34:295-300 (2000) which disclosure is herebyincorporated by reference in its entirety). A complex ‘shutter’ domainis responsible for maintaining the usual, closed state of beta-sheet A(Stein, P E et al., Nature Structural Biology 2:96-113 (1995); Gils, Aet al. Thromb. Haemost. 80:531-41 (1998) which disclosures are herebyincorporated by reference in their entirety).

By virtue of conformational perturbation imposed on the protein by thenovel splicing event, CrypAAT is without proteinase inhibitory function.CrypAAT retains its susceptibility to cleavage by neutrophil elastase,however. It also retains its susceptibility to cleavage by a number ofnon-target proteinases, such as gelatinase B (MMP-9). Unlikeantitrypsin, therefore, CrypAAT functions as a proteinase substrate.Cleavage of CrypAAT by neutrophil elastase and the non-targetproteinases generates a 4 kDa C-terminal fragment of 36 residues, whichon cleavage remains non-covalently bound to the cleaved CrypAAT.

CrypAAT plays a number of diverse physiological roles as a proteinasesubstrate (Janciauskiene, S, Biochimica et Biophysica Acta 1535:221-35(2001) which disclosure is herebly incorporated by reference in itsentirety). Cleaved CrypAAT contributes to the later phase ofpolymorphonuclear leukocyte infiltration and is a potent chemoattractantfor monocytes. The isolated C-terminal fragment of CrypAAT can associatewith extracellular matrix proteins such as collagen and/or laminin-1and, in so doing, play an important role in protecting these proteinsfrom inappropriate enzyme digestion. The C-terminal fragment of CrypAATalso exerts significant effects on cellular lipid catabolism andproinflammatory activation, by activating peroxisomeproliferator-activated receptors (PPARs), transcription factors thatrecently have been proposed to regulate genes for lipid metabolism andproinflammatory proteins.

CrypAAT has also been implicated in several pathologies, namelyatherosclerosis and cancer. The C-terminal cleavage fragment of CrypAATis a component of atherosclerotic plaque, located specifically in thefibrous cap near the necrotic core. CrypAAT plays a role inatherosclerosis as a protease substrate and a reservoir ofphysiologically active peptide degradation products. CrypAAT-positiveadenocarcinomas of colon and lung have a worse prognosis thanCrypAAT-negative ones. Recent studies provide good experimental evidencethat the C-terminal fragment of CrypAAT generated by matrixmetalloproteinases (MMPs) enhances tumor growth and invasiveness invivo.

Contrary to previous dogmas, it is now well established that brain cellscan produce cytokines and chemokines, and can express adhesion moleculesthan enable an in situ inflammatory reaction. Brain ischemia and traumaelicit robust inflammation. The accumulation of neutrophils early afterbrain injury is believed to contribute to the degree of brain tissueloss.

In a preferred embodiment, the present invention provides for anantibody that specifically binds CrypAAT of the present invention.Further preferred is a method for making said antibody wherein saidantibody recognizes a non-conformational or conformational epitope ofCrypAAT.

Further preferred is a method for making said antibody wherein a mouseis immunized with CrypAAT. Further preferred is a method whereinmonoclonal antibodies derived from said mouse are screened for bindingto CrypAAT but not to antitrypsin. Further preferred is a method whereinmonoclonal antibodies derived from said mouse are screened byenzyme-linked immunosorbent assay (ELISA) for binding to CrypAAT but notto antitrypsin. Further preferred is a method wherein said antibody isscreened for the capacity to sterically or allosterically abrogate theprotease susceptibility of CrypAAT. Further preferred is a methodwherein said antibody is screened for the capacity to sterically orallosterically abrogate the neutrophil elastase or gelatinasesusceptibility of CrypAAT. Methods of generating said monoclonalantibody and of establishing its specificity by methods including ELISAare well known to those skilled in the art. Methods of screening saidantibody for the capacity to abrogate the protease susceptibility ofCrypAAT are well known to those skilled in the art and include, but arenot limited to: contacting the antibody with CrypAAT, incubating theantibody-CrypAAT complex with neutrophil elastase or gelatinase, andfollowing proteolytic generation of the 4 kDa carboxyl fragment bydenaturing polyacrylamide gel electrophoresis.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with CrypAAT.Further preferred is a method for using said antibody diagnostically todetermine the basis either for immune dysfunction or forinflammopathology. Further preferred is a method of using said antibodydiagnostically in a sandwich ELISA format to quantitate CrypAAT inplasma or other bodily fluid, including but not restricted to synovialfluid and cerebrospinal fluid, within a pathological context. Furtherpreferred is a method of using said diagnostic assay to determine thelevel of CrypAAT in plasma or other bodily fluid of a patient witheither dysregulated immune function or inflammopathology wherein theimmune dysfunction or inflammopathology is selected from, but notrestricted to, the group consisting of: (a) Rheumatoid arthritis; (b)Atheriosclerosis; (c) Inflammatory bowel disease; (d) Insulin dependentdiabetes mellitus (Type 1 diabetes); (e) Systemic lupus erythematosus;(f) Multiple sclerosis; (g) Psoriasis; (h) Allergic asthma; (i) Acutemyocardial infarction; (j) Septic shock; (k) Reperfusion injury; and (l)Stroke.

In further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to CrypAAT said antibodyhaving the capacity to abrogate the proteolytic susceptibility,including but not restricted to that of neutrophil elastase andgelatinase, of CrypAAT. Further preferred is a method of using saidantibody in contact with CrypAAT as a therapeutic for patients witheither immune dysfunction or inflammopathology. Preferred compositionscomprise said CrypAAT antibody or fragments or derivatives thereof.Preferred formulation of said composition is that compatible with theroute of delivery wherein said route of delivery is selected from, butnot restricted to, the group: (a) Oral; (b) Transdermal; (c) Injection;(d) Buccal; and (e) Aerosol.

In further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to CrypAAT said antibodyhaving the capacity to abrogate the proteolytic susceptibility,including but not restricted to that of neutrophil elastase andgelatinase, susceptibility of CrypAAT. Further preferred is a method forusing said CrypAAT antibody to treat patients with immune dysfunction orinflammopathology. Further preferred is a method of treating saidpatients with said CrypAAT antibody in a method of ameliorating thesymptoms or pathology associated with immune dysfunction orinflammopathology. Said CrypAAT antibody ameliorates the symptoms orpathology associated with immune dysfunction or inflammopathology bysuppressing proteolytic generation of bioactive fragments of CrypAAT,including but not restricted to the 4 kDa carboxyl fragment. Furtherpreferred is a method of delivering to said patients an ameliorativeeffective amount of said CrypAAT antibody. Further preferred is a methodof delivering to said patients an ameliorative effective amount of saidCrypAAT antibody by injection. Further preferred is a method ofdelivering to said patients with immune dysfunction or inflammopathologyan ameliorative effective amount of said CrypAAT antibody wherein saidimmune dysfunction or inflammopathology is selected from, but notrestricted to, the group: (a) Rheumatoid arthritis; (b)Atheriosclerosis; (c) Inflammatory bowel disease; (d) Insulin dependentdiabetes mellitus (Type 1 diabetes); (e) Systemic lupus erythematosus;(f) Psoriasis; (g) Multiple sclerosis; (h) Allergic asthma; (i) Acutemyocardial infarction; (j) Septic shock; (k) Reperfusion injury; and (l)Stroke.

Further preferred is a method of contacting and specifically bindingsaid antibody with CrypAAT in a method of transdermal contact toameliorate the symptoms or pathology of psoriasis. Further preferred arecompositions comprised of said CrypAAT antibody used in methods ofcontacting the psoriatic lesion with an ameliorative effective amount ofsaid CrypAAT antibody by injection or transdermal contact at the site ofthe lesion.

Further preferred is a method of contacting and specifically bindingsaid antibody with rypAAT in a method to ameliorate the symptoms orpathology of allergic asthma. Preferred route of delivery is aerosol.Further preferred are compositions comprised of said CrypAAT antibodyused in methods of contacting asthmatic tissue with an ameliorativeeffective amount of said CrypAAT antibody by aerosol.

Further preferred is a method of contacting and specifically bindingsaid antibody with CrypAAT in a method to ameliorate the symptoms orpathology of allergic rhinitis (hayfever). Preferred route of deliveryis aerosol. Further preferred are compositions comprised of said CrypAATantibody used in methods of contacting inflamed nasal tissue with anameliorative effective amount of said CrypAAT antibody by aerosol.

In a further embodiment, the present invention provides for said CrypAATantibody to be used in a method to suppress acute inflammation. Furtherpreferred is a method to use said CrypAAT antibody to suppressinflammation associated with wound healing. Further preferred arecompositions comprised of said antibody used in methods of contacting awound or injured tissue with an ameliorative effective amount byinjection or transdermal contact at the site of the wound.

In further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to CrypAAT said antibodyhaving the capacity to abrogate the proteolytic susceptibility,including but not restricted to that of neutrophil elastase andgelatinase, susceptibility of CrypAAT. Further preferred is a method oftreating cancer patients with said CrypAAT antibody in a method ofameliorating the symptoms or pathology associated with the cancer. SaidCrypAAT antibody ameliorates the symptoms or pathology associated withcancer (including but not restricted to metastasis and invasiveness) bysuppressing proteolytic generation of bioactive fragments of CrypAAT,including but not restricted to the 4 kDa carboxyl fragment. Furtherpreferred is a method of delivering to said patients an ameliorativeeffective amount of said CrypAAT antibody by said route of delivery.Preferred route of delivery is intravenous or intra-tumoral injection.Further preferred is a method of delivering to said patients with canceran ameliorative effective amount of said CrypAAT antibody wherein saidcancer is selected from, but not restricted to, the group: (a) Melanoma;(b) Breast carcinoma; (c) Lung carcinoma; (d) Colon carcinoma; (e)Hodgkin's lymphoma; (f) Non-Hodgkin's lymphoma; (g) Prostatic carcinoma;(h) Pancreatic carcinoma; (i) Uterine carcinoma; (j) Ovarian carcinoma;(k) Testicular carcinoma; (l) Renal carcinoma; (m) Hepatic carcinoma;and (n) Lung non-small-cell carcinoma.

In a further embodiment, the present invention provides for said CrypAATantibody to be used in a method of preclinical pharmacology in animalmodels of disease, including but not restricted to those of immunedysfunction, inflammopathology, and cancer.

Further preferred is a method in which said CrypAAT antibody is used ina rodent or primate model of human immune dysfunction orinflammopathology to optimize the therapeutic efficacy of said CrypAATantibody. Further preferred is a method in which said CrypAAT antibodyis used in a rodent or primate model of human immune dysfunction orinflammopathology wherein said immune dysfunction or inflammopathologyis selected from but not restricted to the group: (a) Rheumatoidarthritis; Atheriosclerosis; Inflammatory bowel disease; Insulindependent diabetes (Type 1 diabetes); Systemic lupus erythematosus;Psoriasis; Multiple sclerosis; Allergic asthma; Acute myocardialinfarction; Septic shock; Reperfusion injury; and Stroke.

Further preferred is a method in which said CrypAAT antibody is used ina mouse model of human cancer to optimize the therapeutic efficacy ofsaid CrypAAT antibody. Further preferred is a method in which saidCrypAAT antibody is used in a xenogeneic mouse model of human leukemia.Preferred route of delivering said composition comprised of CrypAATantibody includes but is not restricted to intravenous injection andimplanted pump. Further preferred is a method in which said CrypAATantibody is used in a xenogeneic mouse model of human leukemia engraftedwith primary leukemia cells obtained from patients (Dialynas, D P etal., Blood 97:3218-25 (2001) which disclosure is hereby incorporated byreference in its entirety) wherein the leukemia is selected from but notrestricted to the group: (a) Childhood T lymphocyte acute lymphoblasticleukemia (Pediatric T-ALL); (b) Adult T lymphocyte acute lymphoblasticleukemia (Adult T-ALL); (c) B lymphocyte acute lymphoblastic leukemia(B-ALL); (d) Acute myeloid leukemia (AML); (e) Chronic lymphocyticleukemia (CLL); and (f) Multiple myeloma.

In a further embodiment, the present invention provides for the use ofsaid CrypAAT antibody in a method to abrogate proteolytic generation ofthe bioactive 4 kDa fragment of CrypAAT in in vitro cell cultures usinghuman serum. Further preferred is a method of contacting andspecifically binding said CrypAAT antibody to CrypAAT in culture toblock in situ proteolytic generation of the bioactive 4 kDa carboxylfragment from CrypAAT introduced into culture by human serum. Furtherpreferred is a method of contacting and specifically binding saidCrypAAT antibody immobilized on a resin to CrypAAT to deplete CrypAATfrom human serum samples by immunoaffinity chromatography.

In a further embodiment, the present invention provides for thescreening of test compounds for the capacity to specifically bind toCrypAAT and block the proteolytic generation of the 4 kDa carboxylfragment by proteases including but not restricted to neutrophilelastase and gelatinase. Further preferred are said test compounds thatspecifically bind to either a non-conformational or conformational siteon CrypAAT. Further preferred are said test compounds that block saidproteolytic cleavage of CrypAAT either sterically or allosterically.Further preferred is a method of screening said test compounds for thecapacity to block cleavage within the active site of CrypAAT byneutrophil eleastase or non-target proteinase and so generate the 4 kDacarboxyl fragment. Methods of screening said test compound for thecapacity to abrogate the protease susceptibility of CrypAAT are wellknown to those skilled in the art and include, but are not limited to:contacting the test compound with CrypAAT, incubating the testcompound-CrypAAT complex with neutrophil elastase or gelatinase, andfollowing proteolytic generation of the 4 kDa carboxyl fragment bydenaturing polyacrylamide gel electrophoresis.

Preferred formulations of said compound are those selected from, but notrestricted to, formulations amenable to the routes of delivery selectedfrom the group: (a) Oral; (b) Transdermal; (c) Injection; (d) Buccal;and (e) Aerosol.

Compounds found to block the cleavage of CrypAAT within its active siteby elastase or non-target proteinase are used in in vivo and in vitromethods analogous to those described above for CrypAAT antibody.

Protein of SEQ ID NO:54 (Internal Designation Clone789749_(—)182-14-3-0-C12-F)

The cDNA of clone 789749_(—)182-14-3-0-C12-F (SEQ ID NO:53) encodes theprotein of SEQ ID NO:54 comprising the amino acid sequence:

MHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN.

Accordingly it will be appreciated that all characteristics and uses ofpolypeptides of SEQ ID NO:54 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 789749_(—)182-14-3-0-C12-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:53 described throughout the present application alsopertain to the nucleic acids included in Clone789749_(—)182-14-3-0-C12-F. Also preferred are fragments having abiological activity as described therein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:54 encodes Plasminute, a variant of plasminresulting from alternative transcription initiation within theplasminogen gene. Plasminute has novel function as described below.

The terminal event in activation of the human fibrinolytic system isgeneration of the enzyme plasmin, a serine protease possessing a varietyof functional properties, the most notable of which is clearance byproteolytic degradation of fibrin deposits. Plasmin is formed uponactivation of its zymogen, plasminogen, as a result of cleavage of asingle peptide bond. This latter event is catalyzed by serine proteaseswith narrow specificity, termed plasminogen activators. Urokinase-typeplasminogen activator and tissue-type plasminogen activator actdirectly; streptokinase acts indirectly. In its capacity as a serineprotease, plasmin functions to dissolve the fibrin clot. Each of theseplasminogen activators is commercially available and is indicated forthe treatment of acute vascular diseases such a myocardial infarct,stroke, pulmonary embolism, deep vein thrombosis, peripheral arterialocclusion, and other venous thromboses (U.S. Pat. No. 5,753,486; “Humantissue plasminogen activator;” which disclosure is hereby incorporatedby reference in its entirety). Collectively, these diseases account formajor health hazards and risks.

In its capacity as a serine protease, plasmin also plays a role innormal processes involving cell migration in tissue remodeling. In thisregard, plasmin is believed to function in processes in which cellmovement is essential, such as macrophage invasion in inflammation,angiogenesis, and keratinocyte accumulation after wound healing.Furthermore, plasmin has also been strongly implicated as an importantmediator in pathological processes of cell migration that are involvedin tumor cell growth (plasmin can activate growth factors) and invasionof surrounding tissue and, perhaps, metastases. Involvement of plasminin these latter processes is supported by the ability of plasmin todegrade extracellular matrix proteins directly, such as proteoglycans,fibronectin, laminin, and type IV collagen, and/or be indirectlyresponsible for the degradation of matrix proteins through activation ofmetalloprotease zymogens, such as stromolysin and procollagenase. As aresult of degradation of the extracellular matrix, cell migration intosurrounding areas becomes more facile (Castellino, F J in MolecularBasis of Thrombosis and Hemostasis, High, K A & Roberts, H R, editors,New York, pp 495-515 (1995) which disclosure is hereby incorporated byreference in its entirety).

Plasminogen is synthesized by endothelial cells as an 810-residue singlechain glycoprotein, from which is excised a 19-residue signal peptideduring secretion. Plasminogen is converted to plasmin as a result ofactivator-catalyzed cleavage of the Arg561-Val562 peptide bond (numberedfrom the amino-terminal glutamic acid residue of secreted plasminogen).The resulting plasmin contains a heavy chain, originating from the aamino-terminus of plasminogen, doubly disulfide-linked to a light chain.This latter region, containing the carboxy-terminus of plasminogen, ishomologous to serine proteases such as trypsin and elastase. The heavychain of plasmin consists of five repeating triple-disulfide-linkedpeptide regions, about 80 amino acid residues in length, terminedkringles, that are responsible in part for interactions of plasmin withinhibitors (Castellino, F J et al., Ciba Found. Symp. 212:46-60 (1997)which disclosure is hereby incorporated by reference in its entirety).

The gene for human plasminogen spans about 52.5 kilobases of DNA andconsists of 19 exons separated by 18 introns (Petersen, T E et al., J.Biol. Chem. 265:6104-11 (1990) which disclosure is hereby incorporatedby reference in its entirety).

Plasminogens exist in about three different forms in solution, asdetermined by laser light scattering experiments and NMR. Threetruncated forms of plasminogen have been described. Removal of theamino-terminal domain yields a shorter proenzyme (Lys-plasminogen) thatis more efficiently activated than the parent (Glu-plasminogen). Furthercleavage using the enzyme elastase removes the a amino-terminal domainand four kringle domains leaving miniplasminogen. Miniplasminogen isactivatable by urokinase to the enzyme miniplasmin with fibrinolyticactivity equivalent to that of plasmin. The most striking functionaldifference of miniplasmin is its relative resistance to inhibition bythe primary plasmin inhibitor, alpha-2-antiplasmin, probably reflectingthe absence of kringle domain 1, which is thought to facilitate primaryinteraction of plasmin with the inhibitor (Moroz, L A, Blood, 58:97-104(1981) which disclosure is hereby incorporated by reference in itsentirety).

A functionally active human microplasminogen without kringle structureswas produced by incubation of plasminogen with urokinase-free plasmin atalkaline pH. Microplasminogen can be activated by urokinase andstreptokinase to catalytically active microplasmin. Microplasminconsists of two polypeptide chains connected by disulfide bonds: one isthe intact light chain, and the other is a peptide of 31 residues fromthe carboxyl-terminal portion of the heavy chain (Shi, G-Y et al., J.Biol. Chem. 263:17071-5 (1988) which disclosure is hereby incorporatedby reference in its entirety).

It is significant that the formation of plasminogen fragments such asminiplasminogen-like molecules has been observed under somepathophysiological conditions. Of particular note is a report thatsynovial fluid in acute inflammatory arthritis (including rheumatoidarthritis), unlike that of acute non-inflammatory arthritis (includingosteoarthritis), contains low molecular weight fragments of plasminogenwith the properties of miniplasminogen (Moroz, LA et al., ThrombosisResearch 43:417-24 (1986) which disclosure is hereby incorporated byreference in its entirety). Whether neutrophil elastase or othermechanisms are responsible for their generation, the presence ininflamed joints of molecules with properties of miniplasminogenindicates a potential for their participation in inflammatory eventswhere plasmin activity has been implicated, as in the activation ofprocollagenase to collagenase in rheumatoid synovium, but where theinhibitory activity of alpha-2-antiplasmin has been invoked as anobstacle to such a view. However, the ability of molecules such asminiplasmin to escape such inhibition suggests the possibility thatgeneration of miniplasmin might lead to activation of procollagenase, ordestroy joint structural proteins directly.

Plasminute is the product of alternative transcription initiation withinthe plasminogen gene. Transcription initiates within intron N (at least1036 nucleotides upstrcam of exon XV) and proceeds through the remainderof the plasminogen gene (Petersen, T E et al., J. Biol. Chem.265:6104-11 (1990); NCBI Accession No. AL09933.25 which disclosures arehereby incorporated by rererence in their entirety). Splicing occursnormally between transcribed exons XV to XIX. Translation initiateswithin exon XV and is carried out in the plasminogen open reading frame.Plasminute represents the carboxyl-terminal fragment of plasminogencorresponding to amino acids 585 to 791 (numbered from theamino-terminal glutamic acid residue of secreted plasminogen).

Importantly, Plasminute is a variant of plasmin distinguished by thenovel manner in which its protease activity escapes regulation.Plasminute retains the catalytic triad of plasmin (His603, Asp646,Ser741, numbered from the amino-terminal glutamic acid residue ofsecreted plasminogen). Plasminute manifests constitutive proteaseactivity, circumventing the requirement for proteolytic activation byvirtue of its translation initiating downstream of the cleavage siteinvolved in the conversion to plasmin from plasminogen (amino acids561-562 of secreted plasminogen, numbered from the amino terminalglutamic acid residue). In addition, the protease activity of Plasminuteis relatively resistant to inhibition by the primary plasmin inhibitor,alpha-2-antiplasmin, by virtue of its translation initiating downstreamof the plasminogen kringle domains.

In a preferred embodiment, the present invention provides for a methodof contacting Plasminute with a blood clot in patients with acutevascular disease. The advantage of Plasminute over plasminogenactivators is two-fold: 1) it circumvents the necessity to generateplasmin within the patient and therefore is more direct andcontrollable; and 2) it is not immediately neutralized by excessalpha-2-antiplasmin, as is the case for most of the plasmin generatedthrough exogenously administered activator (U.S. Pat. No. 5,753,486;“Human tissue plasminogen activator;” which disclosure is herebyincorporated by reference in its entirety). Preferred compositionscomprise Plasminute. Preferred mode of admistration is intravenousinjection.

In further preferred embodiment, the present invention provides for amethod of contacting Plasminute with a blood clot in patients withdiseases having an etiological basis pointing to either a partial or, insevere cases, total occlusion of a blood vessel by a blood clot—thrombusor thromboembolus. Further preferred is a method of contactingPlasminute with a blood clot in said patients for the purpose ofdissolving said clot. Further preferred are compositions comprised ofPlasminute used in methods of contacting a blood clot with anameliorative effective amount in patients with acute vascular diseasewherein the acute vascular disease is selected from, but not restrictedto, the group consisting of: (a) Myocardial infarct; (b) Stroke; (c)Pulmonary embolism; (d) Deep vein thrombosis; (e) Peripheral arterialocclusion; and (f) Other venous thromboses.

Plasmin plays an important role in wound healing, including recoveryfrom myocardial infarction, skin wounds, and arterial neointimaformation. In the course of myocardial infarction, cardiomyocytes dieand a process that resembles wound healing in, for instance, skin woundsand requiring plasmin occurs (Creemers E, et al., Am. J. Pathol.156:1865-73 (2000) which disclosure is hereby incorporated by referencein its entirety). Specifically with respect to skin wounds, plasmin isrequired for the efficient keratinocyte migration necessary for woundclosure (Romer J et al., Nat. Med. 2:287-92 (1996) which disclosure isincorporated by reference in its entirety). With respect to arterialneointima formation, plasmin is required for migration of smooth musclecells into the necrotic center of the induced arterial wall injury(Carmeliet, P et al., J. Clin. Invest. 99:200-8 (1997) which disclosureis incorporated by reference in its entirety).

In further embodiment, the present invention provides for compositionscomprised of Plasminute used in methods of promoting wound healing.Further preferred are compositions comprised of Plasminute used inmethods of contacting said wound with an ameliorative effective amountwherein the wound is selected from, but not restricted to, the groupconsisting of: (a) Myocardial infarction; (b) Skin wound; and (c)Arterial wall injury.

The compositions and methods for treatment of acute vascular disease andwound healing discussed above are not limited to use in humans, but canhave veterinary applications as well.

Partial digestion of a protein by plasmin is frequently exploited in invitro biochemical analysis of said protein (Bewley, T A, Biochemistry16:209-15 (1977); Nawratil, P et al., J. Biol. Chem. 271:31735-41(1996); Kost, C et al., Eur. J. Biochem. 236:682-8 (1996);Angelloz-Nicoud, P et al., Growth Hormone and IGF Research 8:71-75(1998); Itoh, Y et al., J. Biochem. 128:1017-24 (2000); whichdisclosures are hereby incorporated by reference in their entirety). Forexample, partial digestion by plasmin can be useful in assigningfunction to specific protein domains and in mapping antigenic epitopesonto the protein. Plasminute has utility over plasmin for saidbiochemical analysis in that: 1) production of Plasminute does notrequire proteolytic activation of plasminogen; and 2) the smaller sizeof Plasminute makes it easier to manipulate.

Further preferred are compositions comprised of Plasminute used inmethods of in vitro biochemical analysis of protein, including but notrestricted to the analysis of protein function and antigenicity. Furtherpreferred are compositions comprised of Plasminute used as part of a kitin methods of in vitro biochemical analysis of protein, including butnot restricted to the analysis of protein function and antigenicity.

In a preferred embodiment, the present invention provides for anantibody that specifically binds Plasminute of the present invention.Further preferred is a method of making said antibody wherein saidantibody recognizes a non-conformational or conformational epitope ofPlasminute. Further preferred is a method of making said antibodywherein said antibody neutralizes the serine protease activity ofPlasminute or facilitates the elimination of Plasminute from tissue.

Further preferred is a method wherein a mouse is immunized withPlasminute. Further preferred is a method wherein monoclonal antibodiesfrom said mouse are screened for binding to Plasminute but not toplasmin or plasminogen. Further preferred is a method wherein monoclonalantibodies derived from said mouse are screened by enzyme-linkedimmunosorbent assay (ELISA) for binding to Plasminute but not to plasminor plasminogen. Further preferred is a method wherein monoclonalantibodies from said mouse are screened for binding to Plasminute butnot to plasmin, plasminogen, miniplasmin, miniplasminogen, microplasmin,or microplasminogen. Further preferred is a method wherein monoclonalantibodies derived from said mouse are screened by ELISA for binding toPlasminute but not to plasmin, plasminogen, miniplasmin,miniplasminogen, microplasmin, or microplasminogen. Further preferred isa method wherein said antibody is screened for the capacity tosterically or allosterically neutralize the serine protease activity ofPlasminute. Further preferred is a method of humanizing said monoclonalantibody. Methods of generating said monoclonal antibody and ofestablishing specificity by methods including ELISA are well known tothose skilled in the art. Methods of screening said antibody toneutralize the serine protease activity of Plasminute are well known tothose skilled in the art and include, but are not limited to: contactingthe antibody with Plasminute, incubating the antibody-Plasminute complexwith a substrate of Plasminute, and following proteolytic activation ofthe Plasminute substrate. Methods of humanizing said monoclonal antibodyare well known to those skilled in the art.

The functionality of Plasminute is proinflammatory. Functional fragmentsof plasminogen at least as small as miniplasminogen have been observedin synovial fluid in acute inflammatory arthritis but not in synovialfluid in acute non-inflammatory arthritis (Moroz, L A et al., ThrombosisResearch 43:417-24 (1986) which disclosure is hereby incorporated byreference in its entirety).

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method for using said antibody diagnostically todetermine the basis for inflammopathology. Further preferred is a methodfor using said antibody diagnostically in a sandwich ELISA format todetermine the level of Plasminute in plasma or other bodily fluid,including but not restricted to synovial fluid and cerebrospinal fluid,within a pathological context. Further preferred is a method for usingsaid antibody in a sandwich ELISA format to determine the level ofPlasminute in plasma or other bodily fluid, including but not restrictedto synovial fluid and cerebrospinal fluid, from normal subjects in orderto establish a baseline level of Plasminute. Further preferred is amethod of using said diagnostic assay to determine the level ofPlasminute in plasma or other bodily fluid of a patient withinflammopathology wherein the inflammopathology is selected from, butnot restricted to, the group consisting of: (a) Atheriosclerosis; (b)Inflammatory bowel disease; (c) Insuline dependent diabetes mellitus(Type 1 diabetes); (d) Systemic lupus erythematosus; (e) Multiplesclerosis;Psoriasis; (f) Allergic asthma; (g) Septic shock; and (h)Reperfusion injury.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method for using said antibody diagnostically todetermine the basis for inflammatory arthritis. Further preferred is amethod of using said diagnostic assay to determine the level ofPlasminute in synovial fluid of a patient with acute inflammatoryarthritis (a-d below) or acute non-inflammatory arthritis (e-f below).Plasminute level may be additionally useful is distinguishing the formerfrom the latter (Moroz, L A et al., Thrombosis Research 43:417-24 (1986)which disclosure is hereby incorporated by reference in its entirety).

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method of using said diagnostic assay in saidsandwich ELISA format to determine the level of Plasminute in synovialfluid of a patient with acute inflammatory arthritis or acutenon-inflammatory arthritis. Further preferred is a method of using saiddiagnostic assay to determine the level of Plasminute in synovial fluidof a patient with acute inflammatory arthritis or acute non-inflammatoryarthritis wherein the arthritis is selected from, but not restricted to,the group consisting of: (a) Rheumatoid arthritis; (b) Gout; (c) Septicarthritis; (d) Reiter's syndrome; (e) Osteoarthritis; and (f) Trauma.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method of using said antibody diagnostically inan immunohistochemistry format to determine the level of Plasminute inaffected tissue in a patient presenting with inflammopathology. Furtherpreferred is a method of using said antibody diagnostically in animmunohistochemistry format to determine the level of Plasminute inaffected tissue in a patient presenting with inflammopathology whereinsaid inflammopathology is selected from, but not restricted to, thegroup consisting of: (a) Inflammatory arthritis; (b) Atheriosclerosis;(c) Inflammatory bowel disease; (d) Insuline dependent diabetes mellitus(Type 1 diabetes); (e) Systemic lupus erythematosus; (f) Multiplesclerosis; (g) Psoriasis; (h) Allergic asthma; (i) Septic shock; and (j)Reperfusion injury.

The components of the urokinase plasminogen activator system involved inconversion of plasminogen to plasmin are present in significantly higheramounts in malignant tumors than in normal tissue or benign tumors, andsaid elevated expression is related to poor prognosis for a variety ofpatients diagnosed with tumors including breast, prostate, lung, orcolon cancer (Andreasen, P A et al., Cell. Mol. Life Sci. 57:25-40(2000) which disclosure is hereby incorporated by reference in itsentirety) (discussed in more detail below). The largest data setscorrelating urokinase plasminogen activator level with patient prognosisare available for breast cancer. In the Western world, about one inevery ten women will develop breast cancer. In a significant number ofthese patients, metastatic cells will have spread to the lymph nodes andother tissues by the time their breast tumor is diagnosed. Therefore,following surgery these patients will normally receive some kind ofadditional therapy aimed at reducing their risk of developing secondarycancer.

Even for those patients whose lymph nodes are free of tumor cells (nodenegative), it is still important to know whether they are at high or lowrisk of developing secondary tumors. Measuring the levels of thecomponents of the urokinase plasminogen activator system can assess thisrisk, high levels indicating a high risk of developing metastases andsuggesting that patients should be treated with additional therapy. Justas elevated plasmin generation on engagement of the urokinaseplasminogen activator system indicates high risk for metastases,elevated Plasminute expression by the tumor cells indicates high riskfor metastases. In a preferred embodiment, the present inventionprovides for a method of contacting said antibody and specificallybinding it with Plasminute. Further preferred is a method of using saidantibody diagnostically in an immunohistochemistry format to determinethe level of Plasminute in affected tissue in a patient presenting withcancer. Further preferred is a method of using said antibodydiagnostically in an immunohistochemistry format to determine the levelof Plasminute expressed by tumor cells in a patient presenting withcancer wherein said cancer is selected from, but not restricted to, thegroup consisting of: (a) Melanoma; (b) Squamous cell carcinoma of theskin; (c) Breast carcinoma; (d) Lung small-cell carcinoma; (e) Coloncarcinoma; (f) Hodgkin's lymphoma; (g) Non-Hodgkin's lymphoma; (h)Prostatic carcinoma; (i) Pancreatic carcinoma; (j) Osteosarcoma; (k)Uterine carcinoma; (l) Ovarian carcinoma; (m) Chondrosarcoma; (n)Endometrial cancer; (o) Testicular carcinoma; (p) Renal carcinoma; (q)Hepatic carcinoma; (r) Lung non-small-cell carcinoma; (s) T lymphocyteacute lymphoblastic leukemia (T-ALL); (t) B lymphocyte acutelymphoblastic leukemia (B-ALL); (u) Acute myeloid leukemia (AML); (v)Chronic lymphocytic leukemia (CLL); and (w) Multiple myeloma.

Viral hemorrhagic fevers are a group of diseases caused by viruses fromfour distinct families: filoviruses, arenaviruses, flaviviruses, andbunyaviruses. Virus driven expression of host Plasminute and aconsequential hyperfibrinolysis may be contributory to at least somesaid viral pathologies. Furthermore, measuring Plasminute level may havediagnostic value in distinguishing between viruses in this group or havediagnostic value in distinguishing viruses belonging to this group fromviruses not belonging to this group.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method of using said diagnostic assay in sandwichELISA format to determine the level of Plasminute in plasma or otherbodily fluid in a patient suspected of having viral hemorrhagic fever.Further preferred is a method for using said diagnostic assay todetermine the level of Plasminute in a patient infected by a virus notbelonging to the group causing viral hemorrhagic fever, in order toestablish a baseline level of Plasminute level in said other viralinfection. Further preferred is a method of using said diagnostic assayto determine the level of Plasminute in plasma or other bodily fluid inpatient suspected of having viral hemorrhagic fever when said virus isselected from, but is not restricted to, the group consisting of: (a)Ebola virus; (b) Omsk hemorrhagic fever virus; (c) Junin virus; (d)Marburg virus; (e) Crimean-Congo hemorrhagic fever virus; and (f) Denguefever virus.

The serine protease activity of Plasminute is relatively resistant toinhibition by the primary plasmin inhibitor, alpha-2-antiplasmin, byvirtue of its translation initiating downstream of the plasminogenkringle domains. In in vitro analysis of clinical samples, it isimportant to prevent artifactual proteolysis of the sample ex vivo,including that by plasmin or a derivative thereof. If the samplecontains Plasminute, the current art of using alpha-2-antiplasmin toblock said proteolysis would be inadequate. In this context, saidantibody directed to Plasminute and neutralizing its serine proteaseactivity would have utility over alpha-2-antiplasmin. In a preferredembodiment, the present invention provides for a method of contactingsaid antibody and specifically binding it with Plasminute. Furtherpreferred is a method of using said neutralizing anti-Plasminuteantibody to block ex vivo proteolysis by Plasminute within clinicalsamples.

There is precedent for the expression of two alternatively splicedtranscripts derived from the same gene and encoding functionallydistinct protein isoforms being reciprocally modulated by cytokine. Thisis the case for monocyte expression of CD86, a T lymphocyteco-stimulator molecule, for example. Interferon gamma down-regulatesmonocyte expression of the alternatively spliced transcript encoding atruncated and interfering version of CD86 and up-regulates the splicedtranscript encoding full-length CD86 (Magistrelli, G et al., Biochem.Biophys. Res. Commun. 280:1211-5 (2001) which disclosure is herebyincorporated by reference in its entirety). It is not unreasonable toexpect, therefore, that there may be cytokine modulation oftranscription initiation within a gene leading to alternativetranscripts encoding functionally distinct protein isoforms, as is thecase for the present invention. Identification of said cytokineregulation of alternative transcription initiation within a gene wouldbe expected to have therapeutic value and to lead to a betterunderstanding of disease pathology.

In a preferred embodiment, the present invention provides for a methodof contacting said antibody and specifically binding it with Plasminute.Further preferred is a method of using said antibody to characterizecytokine regulation of Plasminute expression by endothelial cells.Further preferred is a method of using said antibody in said sandwichELISA to characterize cytokine regulation of Plasminute expression byendothelial cells. Further preferred is a method of using said antibodyin said sandwich ELISA to characterize cytokine regulation of Plasminuteexpression by endothelial cells wherein the cytokine is selected from,but not restricted to, the group consisting of: (a) Interferon gamma;(b) Interleukin 17; (c) Interleukin 4; (d) Interleukin 10; (e)Interleukin 13; (f) Interleukin 15; (g) Interleukin 12; (h) Interleukin18; (i) Interleukin 20; (j) Interleukin 21; (k) Interleukin 1 beta; (1)Interleukin 6; (m) Monocyte chemotactic protein 1 (MCP-1); (n) RANTES;(o) IP-10; (p) Vascular endothelial growth factor (VEGF); (q)Transforming growth factor beta; (r) Interleukin 8; and (s) Tumornecrosis factor alpha.

Methods of characterizing cytokine regulation of Plasminute expressionby endothelial cells are well known to those skilled in the art andinclude, but are not limited to: incubation of endothelial cells with orwithout cytokine for 24-48 hours, collection of culture supernatant, anddetermination of Plasminute protein in the culture supernatant bysandwich ELISA.

The transcript encoding Plasminute can be readily distinguished fromthat encoding plasminogen and its derivatives. Further preferredtherefore is a method of directly characterizing cytokine regulation ofPlasminute mRNA expression by endothelial cells. Further preferred is amethod of using polynucleotide comprising Plasminute to determine thelevel of Plasminute mRNA in endothelial cells. Further preferred is amethod of using polynucleotide comprising Plasminute to determine thelevel of Plasminute mRNA in endothelial cells that have been incubatedin the presence or absence of cytokine for 0, 2, 4, 6, 8, 12, or 24hours. Further preferred is a method of using a Plasminute cDNA fragmentencoding 5′-untranslated sequence derived from intron N as a specificprobe in Northern blot analysis of said Plasminute mRNA level. Furtherpreferred is a method of using a primer specified in Plasminute5′-untranslated sequence derived from intron N in conjunction with aprimer specified in Plasminute 3′-untranslated sequence to specificallydetermine said Plasminute mRNA level by reverse transcriptase-polymerasechain reaction (RT-PCR). Methods of carrying out Northern blot analysisor RT-PCR on total or poly(A)+ RNA are well known to those in the art.

The functionality of Plasminute is proinflammatory. In this context, itis significant that functional fragments of plasminogen at least assmall as miniplasminogen have been observed in synovial fluid in acuteinflammatory arthritis but not in synovial fluid in acutenon-inflammatory arthritis (Moroz, L A et al., Thrombosis Research43:417-24 (1986) which disclosure is hereby incorporated by reference inits entirety). Said neutralizing anti-Plasminute antibody would beexpected to have therapeutic value in inflammopathologies in whichPlasminute plays a role.

In its capacity as a serine protease, plasmin plays a role in normalprocesses involving cell migration in tissue remodeling. In this regard,plasmin is believed to function in processes in which cell movement isessential, such as macrophage invasion in inflammation and angiogenesis.Involvement of plasmin in these processes is supported by the ability ofplasmin to degrade extracellular matrix proteins directly, such asproteoglycans, fibronectin, laminin, and type IV collagen, and/or beindirectly responsible for the degradation of matrix proteins throughactivation of metalloprotease zymogens, such as stromolysin andprocollagenase. As a result of degradation of the extracellular matrix,cell migration into surrounding areas becomes more facile (Castellino, FJ in Molecular Basis of Thrombosis and Hemostasis, High, K A & Roberts,H R, editors, New York, pp 495-515 (1995) which disclosure is herebyincorporated by reference in its entirety).

Neovascularization plays a role in a number of diseases, including butnot limited to rheumatoid arthritis (Danis, R P et al., Expert Opin.Pharmacother. 2:395407 (2001) which disclosure is hereby incorporated byreference in its entirety).

In a further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to Plasminute saidantibody having the capacity to neutralize the serine protease activityof Plasminute or to facilitate the elimination of Plasminute fromtissue. Further preferred is a method of using said antibody in contactwith Plasminute as a therapeutic for patients with inflammopathology.Preferred compositions comprise said Plasminute antibody or fragments orderivatives thereof. Preferred formulation of said composition is thatcompatible with the route of delivery wherein said route of delivery isselected from, but not restricted to the group consisting of: (a) Oral;(b) Transdermal; (c) Injection; (d) Buccal; and (e) Aerosol.

In further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to Plasminute saidantibody having the capacity to neutralize the serine protease activityof Plasminute or to facilitate the elimination of Plasminute fromtissue. Further preferred is a method of using said Plasminute antibodyto treat patients with inflammopathology. Further preferred is a methodof using said composition comprised of said Plasminute antibody toameliorate the symptoms or pathology associated with saidinflammopathology. Said Plasminute antibody ameliorates the symptoms orpathology associated with said inflammopathology by blocking theproteolytic remodeling of matrix that is directly or indirectly mediatedby Plasminute and that facilitates the inflammatory process, includingmacrophage invasion, or angiogenesis that is associated with thepathology. Further preferred is a method of delivering to patients withsaid inflammopathology an ameliorative effective amount of saidPlasminute antibody wherein said inflammopathology is selected from, butnot restricted to, the group consisting of: (a) Rheumatoid arthritis;(b) Atheriosclerosis; (c) Inflammatory bowel disease; (d) Insulindependent diabetes mellitus (Type 1 diabetes); (e) Systemic lupuserythematosus; (f) Multiple sclerosis; (g) Psoriasis; (h) Allergicasthma; (i) Septic shock; and (j) Reperfusion injury.

Proliferative diabetic retinopathy (PDR) remains one of the major causesof aquired blindness in developed nations. The hallmark of PDR isneovascularization, abnormal angiogenesis that may ultimately causesevere vitreous cavity bleeding and/or retinal detachment. In a furtherembodiment of the invention, said composition comprised of neutralizinganti-Plasminute antibody is used in a method to treat patients with saidPDR.

In its capacity as a serine protease, plasmin plays a role inpathological processes of cell migration that are involved in tumor cellgrowth and invasion of surrounding tissue and, perhaps, metastases[Andreasen, P A et al., Cell. Mol. Life Sci. 57:2540 (2000), whichdisclosure is hereby incorporated by reference in its entirety].Involvement of plasmin in these processes is supported by the ability ofplasmin to degrade extracellular matrix proteins directly, such asproteoglycans, fibronectin, laminin, and type IV collagen, and/or beindirectly responsible for the degradation of matrix proteins throughactivation of metalloprotease zymogens, such as stromolysin andprocollagenase. As a result of degradation of the extracellular matrix,cell migration into surrounding areas becomes more facile (Castellino, FJ in Molecular Basis of Thrombosis and Hemostasis, High, K A & Roberts,H R, editors, New York, pp 495-515 (1995) which disclosure is herebyincorporated by reference in its entirety).

The urokinase plasminogen activator system, and by implication plasmin,is associated with high risk of tumor invasiveness and metastates[Konno, H et al., Jpn. J. Cancer Res. 92:516-(2001); Fisher, J L et al.,Clin. Cancer Res. 7:1654-60 (2001); Vazquez-Rivera, F et al.,Proceedings of the 11th NCI-EORTC-AACR Symposium, Abstract 294 (2000);Ellrieder, V et al., Annals of Oncology 10, suppl.4, 41-45 (1999);Smolarz, B et al., Med. Sci. Monit. 5:833-7 (1999); Romer, J et al., J.Invest. Dermatol. 116:353-8 (2001); Abe, J et al., Cancer 86:2602-11(1999); Morii, T et al., Anticancer Res. 20(5A):3031-6 (2000); Tecimer,C et al., Gynecol. Oncol. 80:48-55 (2001); Zheng, Q et al., J. CancerRes. Clin. Oncol. 126:641-6 (2000); Swiercz, R et al. Oncol. Rep.8:463-70 (2001); Borgfeldt, C et al. Int. J. Cancer 92:497-502 (2001);He, C et al., J. Cancer Res. Clin. Oncol. 127:180-6 (2001); whichdisclosures are hereby incorporated by reference in their entirety].

In further preferred embodiment, the present invention provides for amethod of contacting and specifically binding to Plasminute saidantibody having the capacity to neutralize the serine protease activityof Plasminute or to facilitate the elimination of Plasminute fromtissue. Further preferred is a method of using said Plasminute antibodyto treat patients with cancer. Further preferred is a method of usingsaid composition comprised of said Plasminute antibody to ameliorate thesymptoms or pathology associated with said cancer. Said Plasminuteantibody ameliorates the symptoms or pathology associated with saidcancer by blocking the proteolytic remodeling of matrix that is directlyor indirectly mediated by Plasminute and that facilitates the invasiveand metastatic processes or angiogenesis that is associated with thepathology. Further preferred is a method of delivering said compositioncomprised of said Plasminute antibody by intravenous injection. Furtherpreferred is a method of delivering to patients with said cancer anameliorative effective amount of said Plasminute antibody wherein saidcancer is selected from, but not restricted to, the group consisting of:(a) Melanoma; (b) Squamous cell carcinoma of the skin; (c) Breastcarcinoma; (d) Lung small-cell carcinoma; (e) Colon carcinoma; (f)Hodgkin's lymphoma; (g) Non-Hodgkin's lymphoma; (h) Prostatic carcinoma;(i) Pancreatic carcinoma; (j) Osteosarcoma; (k) Uterine carcinoma; (m)Ovarian carcinoma; (n) Chondrosarcoma; (o) Endometrial cancer; (p)Testicular carcinoma; (q) Renal carcinoma; (r) Hepatic carcinoma; (s)Lung non-small-cell carcinoma; (t) T lymphocyte acute lymphoblasticleukemia (T-ALL); (u) B lymphocyte acute lymphoblastic leukemia (B-ALL);(v) Acute myeloid leukemia (AML); (w) Chronic lymphocytic leukemia(CLL); and (x) Multiple myeloma.

In a further preferred embodiment, the present invention provides for amethod of screening test compounds for the ability to bind Plasminuteand specifically neutralize the serine protease activity of Plasminute.Further preferred are said test compounds that bind to either anon-conformational or conformational site on Plasminute. Furtherpreferred are test compounds that neutralize said serine proteaseactivity of Plasminute either sterically or allosterically. Furtherpreferred is a method of screening said test compounds for the capacityto neutralize said serine protease activity of Plasminute. Methods ofscreening said test compounds for the capacity to neutralize said serineprotease activity of Plasminute are well known to those skilled in theart and include, but are not limited to: contacting the test compoundwith Plasminute, incubating the test compound-Plasminute complex with asubstrate of Plasminute, and following proteolytic activation of thePlasminute substrate.

Preferred formulations of said compound are those selected from, but notrestricted to, the group consisting of: (a) Oral; (b) Transdermal; (c)Injection; (d) Buccal; and (e) Aerosol.

Said compounds found to bind to and specifically neutralize the serineprotease activity of Plasminute are used in methods analogous to thosedescribed above for neutralizing anti-Plasminute antibody.

Protein of SEQ ID NO:56 (Internal Designation Clone519757_(—)184-4-2-0-F7-F)

The cDNA of clone 519757_(—)184-4-2-0-F7-F (SEQ ID NO:55) encodes thehuman intracellular signaling protein comprising the amino acidsequence:

MLEVSDALGGPGRVPGATAGMNGVDTSLLCDLLQALTFLTRNEILCIHDTFLKLCPPGKYYKEATLTMDQVSSLPALRVNPFRDRICRVFSHKGMFSFEDVLGMASVFSEQACPSLKIEYAFRIYDFNENGFIDEEDLQRIILRLLNSDDMSEDLLMDLTNHVLSESDLDNDNMLSFSEFEHAMAKSPDFMNSFRIHFWGC and shares features with the Calcium andIntegrin-Binding (CIB)-and the DNA-dependent kinase interacting (KIP)protein. It will be appreciated that all characteristics and uses of thepolynucleotides of SEQ ID NO:55 and polypeptides of SEQ ID NO:56,described throughout the present application also pertain to the humancDNA of clone 519757_(—)184-4-2-0-F7-F and polypeptide fragments encodedthereby. Polypeptide fragments having a biological activity describedherein and polynucleotides encoding the same are included in the presentinvention. Related polypeptide sequences included in the presentinvention areMGQCLRYQMHWEDLEEYQALTFLTRNEILCIHDTFLKLCPPGKYYKEATLTMDQVSSLPALRVNPFRDRICRVFSHKGMFSFEDVLGMASVFSEQACPSLKIEYAFRIYDFNENGFIDEEDLQRIILRLLNSDDMSEDLLMDLTNHVLSESDLDNDNMLSFSEFEHAMAKSPDFMYSFRIRFWGC.

The gene of SEQ ID:55 is located on chromosome 2, is ubiquitouslyexpressed, has two EF-hand calcium- and zinc-binding domains, regulatesCa²⁺-dependent dephosphorylation processes such as neuronaltransmission, muscle glycogen metabolism, and lymphocyte activation andis hereby referred to as CALSIGN. CALSIGN stimulates signaling processeswhich lead to platelet aggregation and blood clot formation. It binds tothe cytoplasmic domain of integrins and regulates integrin function inphysiological processes via the fibrinogen receptor (integrinα_(IIb)β₃), which is expressed on platelets, and thereby activatesintegrin for binding to fibrinogen, fibronectin, the von-Willebrandfactor, vitronectin, and thrombospondin; it also binds to the interferon1-receptor and contributes to signal transduction events in platelets,which lead to strong cell-cell adhesion, platelet aggregation, and bloodclot formation. CALSIGN also facilitates immune responses viarestoration of surface antigen expression and T-cell activation inresponse to viral- and bacterial infections and to endogenous factors.Further characteristics of CALSIGN comprise VDJ-recombination in 13-cellmaturation and surface antigen expression on mature B-cells [Naik etal., J. Biol. Chem. 272:4651-4654, 1997; PCT WO 98/14471, 1998; Wu andLieber, Mutat. Res. 385:13-20, 1997; PCT WO 98/31796, 1998; Hynes, Cell69:11-25, 1992; Smyth et al., Blood 81:2827-2843, 1993; U.S. Pat. No.6,093,565, 2000, which references are hereby incorporated in theirentirety].

In a preferred embodiment, CALSIGN or other polypeptides of theinvention are used in a method for tissue regeneration and wound healingafter injuries. Wounds, in particular those occurring in the skin assecond and third degree bums, stasis ulcers, trophic lesions such asdecubitus ulcers, severe cuts and abrasions, which are commonlyresistant to natural healing processes, may be treated with acomposition comprising CALSIGN or other polypeptides included in theinvention, or fragments thereof, in a formulation, which might include agrowth factor such as platelet derived growth factor (PDGF) orconnective tissue growth factor (CTGF), or a wound dressing with asepticproperties such as silver-coated fibers (U.S. Pat. Nos. 6,149,916, 2000;6,187,743, 2001; 6,087,549, 2000), which references are herebyincorporated in their entirety.

The process of wound healing consists of three phases during which theinjured tissue is repaired, regenerated, and new tissue is reorganizedinto a scar. These three phases are classified as: a) an inflammationphase which begins from day 0 to 3 days, b) a cellular proliferationphase from 3 to 12 days, and c) a remodeling phase from 3 days to about6 months. In all three phases, antioxidants play a vital role in thehealing process. In the inflammation phase, inflammatory cells, mostlyneutrophils, enter the site of the wound followed by lymphocytes,monocytes, and later macrophages. The neutrophils that are stimulatedbegin to release proteases and reactive oxygen species into thesurrounding medium with potential adverse effects on both the adjacenttissues and the invading microorganisms. The oxygen species known to bereleased by the neutrophils are superoxide (O.sub.2.sup.-) through theaction of a plasma membrane-bound NADPH oxidase, hydrogen peroxide(H.sub.2 O.sub.2) formed by action of dismutation of O.sub.2.sup.-, andHOCl produced by the action of myeloperoxidase with H.sub.2 O.sub.2.

The proliferative phase consists of laying down new granulation tissue,and the formation of new blood vessels in the injured area. Thefibroblasts, endothelial cells, and epithelial cells migrate in thewound site. These fibroblasts produce the collagen that is necessary forwound repair. Ascorbic acid is crucial in the formation of collagen.Several studies have demonstrated that ascorbic acid was capable ofovercoming the reduced proliferative capacity of elderly dermalfibroblasts, as well as increasing collagen synthesis in elderly cellsby similar degrees as in newborn cells even though the basal levels ofcollagen synthesis are age dependent A decrease of ascorbic acid at theinjury area will decrease the rate of wound healing. Inreepithelialization, epithelial cells migrate from the free edges of thetissue across the wound. This event is succeeded by the proliferation ofepithelial cells at the periphery of the wound. Research has also shownthat reepithelialization is enhanced by the presence of occlusive wounddressings which maintain a moisture barrer. The final phase of woundhealing, which is remodeling, is effected by both the replacement ofgranulation tissue with collagen and elastin fibers and thedevascularization of the granulation tissue. Recent studies have shownthat topical application of antioxidants, especially alpha-tocopherol,reduces scarring and normalizes blood coagulation during therapy.

A particularly effective healing treatment for wounds and skin defectssuch as bums, ulcers and lesions is the application of a medicinaldressing containing as an essential ingredient starch hydrolysate havingDextrose Equivalent of less than about 35. In such wound treatment thestarch hydrolysate produces the formation of a film which is intimatelyadhered to the underlying granulation tissue and which is semi-permeableto gas and fluids and provides an ideal protective cover that willreduce fluid and plasma losses and invasion by pathogenic bacteria. Inaddition, it appears that the starch hydrolysate provides a topical orlocal hyperalimentation, that is local nutrition, providing a gradualrelease of glucose which is particularly effective in nutrition oftissue, both damaged and nascent, which have become relatively isolatedfrom normal blood flow nutrition. The cessation of blood flow to such anischemic lesion can be developed in a slow and gradual form such as inthe case of decubitus ulcers and stasis ulcers, or may take place moreacutely such as in thermo-radiation and chemical burns. In the absenceof nutrition, the rate of fluid delivery of nutrients decreases bringinga progressive impairment in the viability of cells and tissues. Thiseventually leads to degeneration and death of the tissue and cells in acondition known as necrosis. Necrosis is generally accompanied bybacterial, fungal and/or viral contamination. As further pointed out inthe aforementioned patent, treatment of exudative skin wounds with astarch hydrolysate dressing produces a greatly reduced bacteria count ofan infected wound and inhibits infection of an uninfected wound. Inaddition, application of the starch hydrolysate to a wound or ulcerproduces a film or semi-permeable membrane which allows edematous liquidto pass through while proteinaceous material is retained within thebody, allowing reduction in the volume of exudate in relatively cleancondition.

Compositions which enhance and promote the wound healing processcomprise suspensions of CALSIGN, said fibrous protein, collagen, and apolysaccharide such as a glycosaminoglycan, which exhibits chemotaxisfor fibroblasts or endothelial cells; the preferred glycosaminoglycansare said to be heparin, heparan sulfate, or alginate; collagen type I,vitamins such as ascorbic acid (vitamin C) and alpha-tocopherol (vitaminE), and particulate starch hydrolysate are applied on wounds to promotethe formation and growth of healthy granulation tissue. Wound healingprocesses will be significantly improved by multilayer laminate “wounddressings ” comprising alternate layers of silver or silver-coatedfibers and non-metalized fibers, which promote cellular proliferationand comprise antibacterial, antifungal, and analgesic properties (U.S.Pat. No. 6,087,549, 2000). The repair process for even minor breaches orruptures takes a period of time extending from hours and days to weeks;and in some instances, as in ulceration, the breach or rupture maypersist for extended periods of time, i.e., months or even years. At alltimes, be it brief or extended, the potential for invasion by pathogenicorganisms or foreign substances continues until new tissue has beengenerated to fully close the rupture or breach. Because of the danger ofinfections, the customary management of wounds includes an initialthorough cleansing of the affected area to remove any contaminants suchas dirt, cloth particles, or other debris that may introduce pathogenicmaterials. Any hopelessly damaged tissues may be debrided and antisepticmaterials are applied to make the area as sterile as possible. Ifconsidered necessary, sutures may be used to reduce the area of theunderlying tissues and thereby limit the amount of tissue exposed tosubsequent contamination. The healing process is brought about bycomplex biological mechanisms generally involving several groups ofspecial cells and proteins. Leukocytes, such as neutrophils andmacrophages, crown the wound site and digest foreign pathogens anddebris. Such cells also send out chemical signals that marshalfibroblasts in the wound vicinity and ultimately generate connectivestructures, principally, collagen, which make up a major portion of thenew tissues. Endothelial cells generate new blood capillaries that growinto the reconstructed tissue areas where their presence is necessary tosupply nutrients to the newly growing tissue cells and remove catabolicproducts. As the new capillaries grow, the cells on the margin of thewound simultaneously multiply and grow inwardly. The fibrous tissuearising from this cell growth eventually fills the wound cavity with anetwork of interlacing threads of collagen which in due time, arrangethemselves in firm bands and form the permanent new tissue.

Said method for promoting wound healing comprises the steps of:

Applying to the wound a composition of a therapeutically effectiveconcentration of CALSIGN or other polypeptides included in the inventionin an aqueous suspension with bovine collagen type I andalpha-tocopherol in a mixture with starch hydrolysate of a low dextroseequivalent DE, wherein said composition is chemotactic for fibroblastsand endothelial cells. Said bovine collagen is pre-treated to removeextraneous proteinaceous material by various dissolution, precipitationand filtration techniques to provide pure collagenous product.

The composition may be combined with a combination of vitamins such asvitamin C and vitamin E, and with a therapeutically effectiveconcentration of a purified connective tissue growth factor (CTGF), andplatelet derived growth factor (PDGF).

Said aquous suspension is applied repeatedly to the wound during thehealing to effectively promote the healing process

Said aqueous suspension may be combined with said multilaminate silverdressing for the treatment of postoperative wounds

These embodiments also include the production of an antibody againstCALSIGN and other polypeptides of the invention, wherein said antibodiescan be polyclonal or monoclonal. For the production of recombinantCALSIGN, an expression vector and a corresponding cell system will beused, wherein the expression system can be prokaryotic such as E.coli,and eukaryotic such as Baculovirus/insect cells, or mammalian systems aswell-known in the art.

Protein of SEQ ID NO:58 (Internal Designation Clone625004_(—)188-15-4-0-H6-F)

The cDNA of clone (SEQ ID NO:57) encodes the protein of SEQ ID NO:58,comprising the sequence:

MGPPGFKGKTGHPGLPGPKGDCGKPGPPGSTGRPGAEGEPGAMGPQGRPGPPGHVGPPGPPGQPGPAGISAVGLKGDRGATGERGLAGLPGQPGPPGPQGPPGYGKMGATGMGQQGIPGIPGPPGPMGQPGKAGHCNPSDCFGAMPMEQQYPPMKTMKGPFG.

Accordingly, it will be appreciated that all characteristics and uses ofpolypeptides of SEQ ID NO:58 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in clone 625004_(—)188-15-4-0-H6-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:57 described throughout the present application alsopertain to the nucleic acids included in clone625004_(—)188-15-4-0-H6-F. Also preferred are fragments having abiological activity as described herein and the polynucleotides encodingthe fragments.

The cDNA of SEQ ID NO:57 is a novel splice variant of the human alpha 1type XVI collagen gene (GB M92642. 1) located on chromosome 1,specifically in the p34-35 region. The cDNA clone of SEQ ID NO:57encodes an open reading frame of 489 nucleotides. Whereas the nativeform of human alpha 1 type XVI collagen possess 71 exons encoding a 1603amino-acid protein, the cDNA of SEQ ID NO:57 contains 14 exons andencodes a 163 amino-acid protein of SEQ ID NO:58. The present proteinrepresents the first described variant of the human alpha 1 type XVIcollagen, named vCOL16A1. The present protein contains two collagentriple helix repeat domains (positions 11-70 and 73-131).

Collagens represent a large family of structurally related proteins thatto date includes more than 20 collagen types. These proteins constitutethe major extracellular matrix components of connective tissues and playa dominant role in maintaining the structural integrity of varioustissues and also have a number of other important functions. Collagenscan be divided into two major classes: the fibril-forming collagens andthe non-fibril-forming collagens; the latter class includes a subgroupnamed the fibril-associated collagens with interrupted triple helices(FACIT). The human alpha 1 type XVI collagen exhibits most of thecharacteristics of the proteins of the non-fibril-forming collagenclass.

In one embodiment, the protein of the invention or fragment thereofprovide an in vitro assay to test the specific activity of variousproteases which degrade or denature collagen, such as collagenases andmany others. Methods to assess the activity of such proteases includethe steps of contacting the protease to be tested with the presentprotein, and detecting the amount of proteolytic cleavage of the presentprotein that occurs.

Since collagen fibrils are often heterogenous structures containing morethan one collagen type, the present invention provides a method todetermine the types of collagen present in a tissue or biologicalsample. For example, the collagen composition of a diseased tissue canbe determined by isolating the present protein under conditions that donot disrupt protein-protein interactions, and determining the identityof proteins associated with the present protein. Such associatedproteins can be identified by any standard method including, but notlimited to, immunoprecipitation and immuno-affinity columns.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Such animals are useful for a number ofpurposes, for example because they represent an in vivo assay method fortesting candidate molecules potentially useful for the treatment ofvarious pathophysiological aspects of diseases associated with abnormalcollagen metabolism specifically related to the activity of the presentprotein. Study of the phenotype of such models can also allow theidentification of additional human equivalent diseases caused by orlinked with collagen mutations. These animals can be generated with anymethod of targeting overexpression or inactivation of the presentprotein. In one such embodiment, purified forms of the present proteinare injected into the joints of an animal, or the protein isrecombinantly expressed in the joints, to provoke “collagen inducedarthritis” in the joints, a well known model for arthritis. Such modelsare extremely useful, e.g. in the assessment of candidate therapies anddrugs for the treatment of arthritis and other inflammatory diseases andconditions.

In other embodiment, the protein of the invention or fragment thereof isused to diagnose diseases or disorders associated with abnormalities ofthe metabolism of collagen. Examples of such diseases and disordersinclude, but are not limited to, hereditary nephritis of Alport's typedue to a defect in collagen assembly that lead to progressive renalfailure, disorders of bone tissue comprising osteoporosis, Paget'sdisease, disorders of cartilage tissue occurring in arthritis (such asosteo-arthritis and rheumatoid arthritis), disorders of thecardiovascular system prominent in atherosclerosis, hypertension,myocardial infarction and hypertrophy. This method includes the steps ofcontacting a biological sample obtained from an individual suspected ofsuffering from the disease or condition, or at risk of developing thedisease or condition, with a compound capable of selectively binding thepresent protein or nucleic acids, e.g. a polyclonal or monoclonalantibody or any immunologically active fragment thereof, a nucleic acidprobe, etc., and detecting the level, spatial distribution, or any otherdetectable property of the present protein in the sample, where adifference in the level, spatial distribution, or other property in thesample relative to in a control sample indicates the presence of thedisease or disorder, or of a propensity for developing the disease ordisorder.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment of diseases andconditions associated with collagen matrix destruction, including forwound treatment, including fractures. Such methods comprise theadministration of a therapeutically-effective amount of the presentprotein to a patient suffering from the disease or condition.Preferably, the protein is administered directly to the site of collagenmatrix destruction. The methods and compositions can also be used in,for example, the restoration of surgically induced wounds, or for thecorrection of physiological malfunction, for example to control urinaryincontinence and more specifically for intrinsic sphincter deficiency.In such methods, the present protein can be administered byperi-urethral injection to reduce lumen aperture. These compositions cancomprise the protein of the invention, and, optionally, one or moreother types of collagen, collagen derivatives, or any other compound ofinterest. All of these components may be either obtained from naturalsources or produced by recombinant genetic engineering techniques and/orchemical modification.

Since aberrant degradation of collagen is an indication of disorders ofconnective tissues, another embodiment the present invention is toprovide an assay for the monitoring of collagen degradation in vivo. Theinvention thus includes test kits useful for the quantification in abiological sample of the amount of collagen fragment derived from thedegradation of collagen, i.e. the degradation of the present protein.The kits comprise at least one immunological binding partner, e.g. amonoclonal or polyclonal antibody specific for a peptide derived fromthe degradation of the present protein or the intact present protein andcoupled to detectable markers. Collagen degradation can be measuredeffectively in plasma, serum or blood by any suitable method, includingimmunoassays. Thus, the condition of a subject can be monitoredcontinuously and the quantified amount of collagen fragments measured inthe pathological sample can be compared with the amount quantified in abiological sample of a normal individual.

In this embodiment, the application of such assays can be used tomonitor the progress of therapy administered to treat these or otherconditions. Further, the assays can be used as a measure of toxicity,since the administration of toxic substances often results in tissuedegradation. It can also be used during clinical testing of new drugs toassess the impact of these drugs on collagen metabolism. Thus the assaysmay be applied in any situation wherein the metabolic condition ofcollagen tissues can be used as an index of the condition, treatment, oreffect of substances directly administered to the subject or to whichthe subject is exposed in the environment.

Also in this embodiment, the present invention provides a method ofdetecting the presence and/or monitoring the metastatic progress of amalignancy. Indeed, metastatic potential can be influenced bothpositively and negatively by a variety of cell surface adhesivemolecules that act both independently and in concert with connectivetissue elements such as collagen, allowing subsequent growth of tumorcells at secondary sites in particular tissues. The invention thusincludes test kits useful for quantify the amount of the present proteinor any specifically associated collagen type in a biological samplecomprising the steps of contacting the biological sample with a specificmonoclonal or polyclonal antibody specific for the present protein orany specifically associated collagen type, and coupled to detectablemarkers. Thus, the condition of a patient can be monitored continuouslyand the quantified amount of such proteins measured in the pathologicalsample can be compared with the amount quantified in a biological sampleof a normal individual or with the previous analysis of the samepatient.

Excessive production and deposition of collagen leads to fibrosis andthereby impairs the normal functioning of the affected organ andtissues. There are numerous examples of fibrosis, including theformation of scar tissue following a heart attack, which impairs theability of the heart to pump. Diabetes frequently causes damage/scarringin the kidneys which leads to a progressive loss of kidney function.Even after surgery, scar tissue can form between internal organs causingcontracture, pain, and in some cases, infertility. Thus, the presentinvention provides a method to inhibit collagen accumulation,specifically the accumulation of the present protein, and thereby toavoid delayed healing. The level of the present protein can be inhibitedor decreased using any of a number of methods, including using antisensemolecules or ribozymes, or alternatively the activity of the presentprotein can be inhibited using direct or indirect inhibitor molecules orantagonistic antibodies directed against the present protein. Theinhibition of the expression or the activity of the present protein isalso useful in the treatment of acute fibrosis (in response to variousforms of trauma including injuries, infections, surgery, burns,radiation, chemotherapy treatments) or in the treatment of chronicfibrosis of the most commonly affected organs (heart, liver, kidney,lung, eye and skin), e.g. induced by viral infection, diabetes,hypertension or other chronic conditions.

In another embodiment, the invention is useful for preparing cosmeticcompositions such as skin creams with anti-wrinkle activity. Cosmeticapplications also include the use of the present invention as a dermalimplant to increase tissue size by injections of collagenous suspensionsfollowing eyebrow uplift, for lip augmentation and to rectify facialdefects, frown lines and acne scars. The present protein can be used asan injectible biomaterial as a dermal implant to increase tissue sizefor cosmetic (wrinkle reduction). The protein of the invention is heldto be an ideal biomaterial due to its ability to persist in the bodylong enough to carry out its specific role without developing a foreignbody response that could lead to the premature rejection or overallfailure of the biomaterial. These compositions can comprise purifiedforms of the present protein and, optionally, one or more other types ofcollagen or collagen derivatives. All of these components may be eitherobtained from natural sources or produced by recombinant geneticengineering techniques and/or chemical modification.

The present invention can also be used in a variety of applications as afood source. Since the transmission risks of bovine spongiformencephalopathy to humans from various commonly used bovine derivedproducts, such as bovine collagen, are still unclear, there is a needfor alternative products to replace bovine derived products. Thus,another advantage of the present invention is derived from the fact thatit is a human collagen rather than an animal-derived collagen. It isuseful for making a casing for food products that are usually sausages,but the present invention can also be applied to any type of materialincluding animal meat, fish meat, shellfish, and fish eggs, such assalmon roe, cheese, noodles. In addition, the present protein can beused as the binder element instead of caseins, which have beenconsidered in the art to be indispensable for obtaining satisfactorybinding strength in bound food. Thus, consumers who are allergic tothese proteins can enjoy the bound food prepared containing the presentinvention without the fear of having an allergic reaction. The use ofthe present invention is also attractive for pet food, for example dogsor cats, and can be even more so if it is combined with solid productsconventionally used in animal nutrition, for example pieces of meat orfish, and/or extruded cereals and/or extruded proteins. In a suchembodiment, the present invention can be deliverable as a mixture,including, but not limited to, in a fluidized state, as a mixture in agel state, in a freeze-dried state, or in a salt-precipitated state.

In another embodiment, the present protein can be used as a biomaterialfor tissue engineering, to regenerate or replace damaged tissues. Thepresent invention thus provides various clinical applications for thegeneration of tissues or organs unable to repair or regeneratethemselves. It can be used, for example, to promote bone regeneration,to repair tendons, ligaments or cartilage, to generate blood vessels orheart valves, to create dental implants, but also in burn injuries, fordermal replacement in chronically unstable scars, after skin loss forhereditary, traumatic or oncological reasons, or for cornealreconstruction (see, e.g. Atala, (2000) J Endourol February;14(1):49-57; Schwartzmann (2000) Implant Dent 9(1):63-6; Machens et al.(2000) Cells Tissues Organs 167(2-3):88-94; the disclosures of which arehereby incorporated by reference in their entireties). The presentinvention is suited to the culturing of three-dimensional mammaliantissues for purposes including transplantation or implantation in vivo,and as the primary component of an extracorporeal organ assist device.Methods are also provided involving stem cells, for examplepluripotential cells, which can differentiate into various tissue types(muscle, cartilage, skin, bone, etc) when stimulated by an appropriateenvironment, e.g. comprising the present protein. For example, stemcells can be expanded in vitro and suspended in collagen gel matrices toform composites. The resulting composites will be implanted in a gapdefect as a graft, which after remodeling in vivo, becomes populatedwith host cells and recapitulates normal functional architecture. Inthis embodiment, these substitutes can also serve as in vitro models fortoxicology testing to better understand the response and healingmechanisms in human tissues.

Protein of SEQ ID NO:60 (Internal Designation Clone422353_(—)145-11-3-0-E7-F)

The cDNA of SEQ ID NO:59 encodes the protein of SEQ ID NO:60, comprisingthe sequence:

MCFPKVLSDDMKKLKARMHQAIERFYDKMQNAESGRGQVMSSLAELEDDFKEGYLETVAAYYEEQHPELTPLLEKERDGLRCRGNRSPVPDVEDPATEEPGESFCDKVMRWFQAMLQRLQTWWHGVLAWVKEKVVALVHAVQALWKQFQSFCCSLSELFMSSFQSYGAPRGDKEELTPQKCSEPQSSK. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptide of SEQ ID NO:60 describedthroughout the present application also pertain to the polypeptideencoded by the nucleic acids included in clone422353_(—)145-11-3-0-E7-F. In addition, it will be appreciated that allcharacteristics and uses of the nucleic acid of SEQ ID NO:59 describedthroughout the present application also pertain to the nucleic acidsincluded in clone 422353_(—)145-11-3-0-E7-F. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:59, SEQID NO:60, and Clone 422353_(—)145-11-3-0-E7-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

The protein of SEQ ID NO:60 (NK5) is a novel splice variant of the humanNatural Killer cells protein 4 precursor (NK4) (Genbank accession numberM59807). NK5 is a 188-amino-acid-long protein that displays an RGDcell-attachment sequence from positions 170 to 172. An epitope, locatedfrom positions 163 to 187, overlaps this RGD motif. NK5 displays aputative trans-membrane domain from positions 148 to 168. Contrarily Incontrast to NK4, NK5 displays no signal peptide. The NK4 cDNA contains 6exons (Bernot et alet al., Genomics 50:147-60 (1998)), whereas the NK5cDNA contains 7 exons. Exons 1 and 2 are identical for NK4 and NK5, andexons 5, 6 and 7 of NK5 are identical to exons 4, 5 and 6 of NK4. Exon 3of NK5 is shorter than exon 3 of NK4, and exon 4 is unique for NK5.

NK4 gene expression is ubiquitous (Bernot et al., Genomics 50:147-60(1998)). Nevertheless, its expression is greatly increased inmitogen-activated T cells and in IL-2-activated Natural Killer cells(Dahl et al, J. Immun. 148:597-603 (1992)).

Natural killer (NK) cells and T cells provide anti-infectious,anti-neoplastic, and immunomodulatory function effected by both cytokineproduction and direct cellular cytotoxicity. In particular, NK cellsplay a primary role in preventing and removing cancer cells in the body,removing many types of viruses (including herpes and measles) and havebeen found to be present at low levels in women with endometriosis.Moreover, in addition to these overtly immuno-protective functions, NKcells also mediate a variety of homeostatic functions, particularly inthe regulation of haematopoesis and they may have an important role toplay in the maintenance and development of placentation. The behaviourof NK and T cells in these various situations is regulated by a largenumber of distinct receptors that transmit positive and negativesignals. Resting NK and T cells express a number of surface moleculeswhich, when stimulated, can activate the cytotoxic mechanism. Thebalance of these signals determines whether an NK or T cell does nothingor is activated to proliferate, kill or secrete a wide range ofcytokines and chemokines. More particularely, IL-2 activates manyNK-cell functions, including baseline or “natural” anti-tumorcytotoxicity, antibody-dependent cellular cytotoxicity (ADCC),proliferation, and cytokine production (Trinchieri, Adv. Immunol.47:187-376 (1989)), and IL-2-activated NK cells display a broaderspectrum of reactivity against human and murine tumor target cells. TheRGD motif, which is found in a number of proteins, has been shown toplay a role in cell adhesion. It was shown that anchorage of NK cells isnecessary for full activation (Li et alet al, J Immunother 20:123-30(1997)), and that long term-activated NK cells acquire new adhesiveproperties. This suggests a central role for RGD recognition in theregulation of immune responses.

The expression of the NK5 gene is greatly increased in IL2-activated NKcells and in mitogen mitogen-activated T cells, and thus likely plays animportant role in lymphocyte activation. In particular, NK5 is believedto play a role in the new adhesive properties that are acquired byactivated lymphocytes. As NK5 does not display a signal peptide, NK5likely plays a distinct role from NK4 in this process.

An embodiment of the present invention relates to methods of using NK5or fragment thereof as a marker to selectively detect and/or quantifyactivated T cells and/or activated NK cells. Any method of detecting thepresence, level, or activity of NK5 can be used in such methods. Forexample, the protein of the invention or fragment thereof may be used togenerate specific antibodies using standard methods, and the antibodiescan be used to detect the level of the present protein in a NK cell or aT cell, wherein a detection of a higher level of the present protein inthe cell compared to a control level representative of a resting T cellor NK cell indicates that the cell is activated. Preferably, theantibodies are either directly or indirectly labeled, and bind morespecifically to NK5 than to related proteins such as NK4. Alternatively,the nucleic acid of the invention or fragment thereof may be used tosynthesize specific probes using any technique known to those skilled inthe art. Such antibodies and/or probes may then be used in assays anddiagnostic kits for the detection and/or quantification of activated Tcells and/or activated NK cells in, e.g., bodily fluids, in tissuesamples and in mammalian cell cultures.

In a preferred embodiment, such methods of detecting the polypeptides orpolynucleotides of the invention, e.g. using specific antibodies and/orprobes, can be used to measure the effect of a test compound on T celland/or NK cell activity in mammalian cell cultures. In another preferredembodiment, such methods can be used to monitor the effects of atreatment aiming to increase or decrease T cell and/or NK cell activityin a patient, or to detect the beginning of a graft rejection reactionin a patient.

Another embodiment of the invention relates to compositions and methodsfor inhibiting the expression or activity of NK5 in a patient for thetreatment or prevention of diseases and disorders caused as a result ofT cell and/or NK cell activation. The inhibition and/or reduction of Tcell and/or NK cell activation can be achieved using any suitablemethod, e.g. through the administration of a therapeutically effectiveamount of an antibody that specifically recognizes NK5 or fragmentthereof to a patient. Preferably, the antibody recognizes the epitopeoverlapping the RGD domain. The antibody can be administered alone or incombination with one or more agent known in the art, e.g. otherimmuno-suppressive agents. Administration of the antibody can be donefollowing any method known in the art, including those described in U.S.Pat. No. 5,817,311, which disclosure is hereby incorporated by referencein its entirety. Other inhibitors of NK5 expression or activity whichcan be used include, but are not limited to, antisense molecules,ribozymes, dominant negative forms of NK5, and compounds that decreasethe activity or expression of NK5 in a cell. Such compounds can bereadily identified, e.g. by screening test agents against T cells ornatural killer cells expressing NK5, or capable of expressing NK5, anddetecting the ability of the test agents to inhibit natural killer cellor T cell activation, or to diminish the level of NK5 expression.Diseases and disorders caused as a result of T cell and/or NK cellactivation include, but are not limited to, allergy and asthma, and themethods can also be used in treatments for preventing and/or inhibitingon-going immune responses. More particularly, such treatments can beused to prevent, or inhibit, or reduce in severity graft rejection, orinduce tolerance to graft transplantation. Such transplantation may byway of example include, but not be limited to, transplantation of cells,bone marrow, tissue, solid-organ, bone, etc. Such treatments can also beused to prevent or reduce in severity graft versus host diseases andautoimmune diseases, which by way of example include but are not limitedto rheumatoid arthritis, systemic lupus, multiple sclerosis,insulin-dependent diabetes, hepatitis, rheumatoid arthritis, Gravesdisease, etc.

Another embodiment of the invention relates to the activation and/orprevention of inactivation of NK and/or T cells, based on compositionsand methods containing, e.g., NK5 or fragment thereof, a polynucleotideencoding the protein, or a compound that increases the expression oractivity of NK5. Such compounds can be readily identified, e.g. byscreening test agents against T cells or natural killer cells expressingNK5, or capable of expressing NK5, and detecting the ability of the testagents to enhance natural killer cell or T cell activation, or toincrease the level of NK5 expression. Diseases and disorders that may betreated and/or reduced in severity by T cell and/or NK cell activationinclude but are not limited to tumors, viral infections, inflammation,or conditions associated with impaired immunity, bacterial infections,hepatic dysfunction, liver regeneration, haematopoesis and maintenanceand development of placentation. More particularity, such treatments canbe used to treat proliferative disorders (including various forms ofcancer such as leukemias, lymphomas, sarcomas, melanomas, adenomas,carcinomas of solid tissue, hypoxic tumors, squamous cell carcinomas,genitourinary cancers, hematopoietic cancers, head and neck cancers, andnervous system cancers, benign lesions such as papillomas,atherosclerosis, angiogenesis), viral infections (in particular HBV,HCV, HIV, hepatitis, measles and herpes viruses infections, as well asother viral-induced infections), and other various immune deficiencies.These immune deficiencies may be genetic (e. g. rheumatoid and osteoarthritis and severe combined immunodeficiency (SCID)) or be caused byvarious bacterial or fungal infections (e.g. infections by mycobacteria,Leishmania spp., malaria spp. and candidiasis). Of course, NK5 may alsobe useful where a boost to the immune system generally may be desirable,i.e., in radiation therapy or chemotherapy when treating the cancer. NK5or fragment thereof can be administered alone or in combination withother known agents capable of activating NK and/or T cells, such asmethods described in U.S. Pat. No. 6,245,563 and in U.S. Pat. No.6,197,302, which disclosures are hereby incorporated by reference intheir entireties.

Protein of SEQ ID NO:62 (Internal Designation Clone500715621_(—)204-15-3-0-C6-F)

The cDNA of Clone 500715621_(—)204-15-3-0-C6-F (SEQ ID NO:61) encodesthe 202 amino acid long polypeptide of SEQ ID NO:62 comprising the aminoacid sequence:

MELWGAYLLLCLFSLLTQVTTEPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVCLKGTKVHMKCFLAFTQTKTFHESSEDCISRGGTLSTPQTGSENDALYEYLRQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLPYICQFGIV. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:62 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone500715621_(—)204-15-3-0-C6-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:61described throughout the present application also pertain to the nucleicacids included in Clone 500715621_(—)204-15-3-0-C6-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:61, SEQ ID NO:62, and Clone 500715621_(—)204-15-3-0-C6-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:62 represents a new variant form of the humantetranectin precursor polypeptide (Swissprot entry P05452), harboring anamino acid substitution at position 94 which replaces an alanine residueby a serine residue. The protein of the SEQ ID NO:62 is a 202 amino acidlong polypeptide comprising a 21 amino acid signal peptide followed by a181 amino acid sequence corresponding to a mature polypeptide of theinvention, Plasminogen carrier protein (PLCP).

PLCP is a 68 kilodalton homotrimeric plasminogen-binding protein presentin plasma. In addition to plasminogen, PLCP binds calcium as well as anumber of sulphated polysaccharides including heparin, chondroitin andfucoidan. It also binds Apoliprotein A and fibrin.

In terms of primary and tertiary structure, the protein is related tothe family of Ca(2+)-binding C-type lectins, proteins that bind a widediversity of compounds, including carbohydrates, lipids and proteins.

The protein is encoded by three exons corresponding to three functionaldomains. Exon 3 (nt367 to nt771 on SEQ ID NO:61) encodes the long-formC-type Lectin domain (aa77 to aa198 on SEQ ID NO:62), also termed thecarbohydrate recognition domain (CRD), which is involved in Ca(2+) andplasminogen binding. Exon 2 (nt268 to nt366) encodes an alpha-helixdomain that governs the trimerization of PLCP oligomers by assemblinginto a triple helical coiled-coil structural element. Finally, residuesencoded by exon1 (nt13 to nt267), but not the CRD, bind heparin,suggesting a specific role for this domain in sulphated carbohydrateligand binding (Lorentsen et al. 2000, Biochem. J. 347, 83-87 whichdisclosure is hereby incorporated by reference in its entirety).

PLCP binds plasminogen via its CRD through a specific interaction withthe fourth kringle domain of plasminogen, and binding has been reportedto facilitate the proteolytic activation of plasminogen to plasmin bythe tissue-type plasminogen activator. Because plasminogen activation isinvolved in a variety of extracellular proteolytic events includingfibrinolysis, cell migration, angiogenesis, tumor cell invasion,inflammation, wound healing, and tissue remodeling, PLCP is useful inthe modulation of these biological processes.

The present protein is isolated from human blood, but is also found tobe deposited in the extracellular matrix of various tissues. Inparticular, PLCP is deposited in the tumor surrounding stroma of breast,colon, and ovarian tumors and is found to co-localise withplasmin/plasminogen at the invasive front of cutaneous melanoma lesions,whereas little or no PLCP is found in the corresponding normal tissues.Plasma PLCP level is reduced in cancer patients, and PLCP is useful as aprognostic marker for the diagnosis of certain types of cancer.

Preferred PLCP polypeptides for uses in the methods described belowinclude the polypeptides comprising the amino sequence of:

EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVCLKGTKVHMKCFLAFTQTKTFHESSEDCISRGGTLSTPQTGSENDALYEYLRQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLPYICQF GIV;

A polypeptide comprising the amino acid sequence of:

VCLKGTKVHMKCFLAFTQTKTFHESSEDCISRGGTLSTPQTGSENDALYEYLRQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLPYICQFGIV;

A polypeptide comprising the amino acid sequence of:

VHMKCFLAFTQTKTFHESSEDCISRGGTLSTPQTGSENDALYEYLRQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLP YICQ

In one embodiment, the cDNA of SEQ ID NO:61 bearing a G to Tsubstitution at position 438, which replaces an alanine residue by aserine at position 94 of SEQ ID NO:62, is used for DNA genotyping.Indeed genotyping this locus could be of interest in DNA fingerprintingfor paternity studies or forensic analyses. It could also be used forgenetic association studies, especially in pathologies relating tocoagulation disorders.

In another embodiment, the polynucleotide sequence of the invention isused in pharmacogenomic applications in order to aid in the choice ofthe ideal drug (e.g. a coagulation or anticoagulation drug), or dosageof a drug, for the treatment of a condition or disease in a patient. Forexample, in one embodiment, the invention provides a method ofgenotyping the patient to determine the identity of the nucleotideencoding the amino acid at position 438 of SEQ ID NO:62, andadministering to the patient a drug or a dosage of the drug that hasbeen established to be preferentially efficacious in those with a serineresidue at position 438 (e.g. because of preferential binding of thedrug to the isoform of the protein with a serine at that position). Inanother embodiment, the patient is genotyped for the nucleotide encodingamino acid position 438, and a drug is determined to be not desirablyadministered to the patient, e.g. because side effects are known to beassociated with the administration of the drug to individuals with aserine at position 438.

In another embodiment, the present protein is used to copurifyplasminogen from a biological sample, preferably from a liver cellextract. This is achieved using any method, a large number of which areknown in the art. For example, plasminogen is purified using affinitycolumn chromatography with the protein of SEQ ID NO:62 or bycoimmunopurification using a monoclonal or polyclonal antibody thatspecifically binds the protein of the invention. Purified plasminogen isuseful for many purposes, including for the preparation of therapeuticfibrinolytic compositions.

In a further embodiment, the present protein provides a method to purifya protein harboring one or more kringle domains from a cellular extract,the method comprising using a fragment of the present protein retainingan intact CRD domain, preferably a fragment restricted to the CRD domainitself, to purify the kringle domain-containing protein, e.g. using amethod such as affinity chromatography. Preferably, the protein to bepurified is selected from the group consisting of plasminogen,angiostatin, thrombin, Hepatocyte Growth Factor, Macrophage StimulatingProtein and apolipoprotein a. The protein to be purified using thepresent method is derived from any source, e.g. protein expressed invitro using an invertebrate, yeast or bacterial heterologous expressionsystem.

In another embodiment, the present protein provides a method todetermine the localization of plasminogen in vivo or ex vivo. In onesuch method, a tissue section is contacted with a labeled protein of SEQID NO:62, and the labeling in the tissue section is detected.Plasminogen can also be detected directly from crude cell or tissueextracts using the protein of the invention. Methods for labelingproteins are well known in the art, any of which is used in the presentinvention.

In another embodiment, the protein of SEQ ID NO:62 is used to determinecirculating levels of plasminogen in the blood of an individual, themethod comprising obtaining a blood sample from the individual, usingthe protein of the invention to copurify plasminogen from the bloodsample (e.g. by affinity column chromatography), and measuring the levelof plasminogen in the sample using methods well known in the art, forexample Elisa, western blot or radioinmmunoessay (RIA). Determiningplasminogen levels in circulating blood could be of special interest forthe monitoring of patients with diseases associated with impairedcoagulation or fibrinolysis.

In another embodiment, the present protein is used as a diagnostic orpronostic marker for breast cancer, ovarian cancer, colon or colorectalcancers, the method comprising contacting a blood sample from a patient,preferably a serum sample, with an antibody directed to the presentprotein, and determining the level of PLCP in the sample compared to acontrol level representative of a healthy patient, wherein a lower levelof PLCP in the patient sample relative to the control level indicatesthat the patient has the disease, is at an elevated risk of developingthe disease, or has a worse prognosis that a patient with normal levelsof the protein. The antibody used is either monoclonal or polyclonal andis labeled directly or indirectly for quantification of immune complexesby methods well known to those skilled in the art.

In another embodiment, the present protein provides a transgenic animal,preferably a mammal, more preferably a rodent, with impairedfibrinolytic activity due to no or reduced expression of the protein ofSEQ ID NO:62. Such transgenic animals provide a powerful model in whichto study pathologies associated with defective fibrinolysis, especiallyfibrosis and thrombosis. In addition, such animal is used to screencandidate molecules for the ability to inhibit coagulation or fibrosis.

Transgenic animals with reduced or eliminated PLCP expresssion oractivity is obtained using any of a number of ways, including by PLCPgene knock-out, for example in the mouse, using DNA microinjection intofertilized eggs or transfection of embryonic stem cells. Alternatively,low level expression of the present protein is achieved using antisensmethods, e.g., by placing the reverse nucleotide sequence encoding theprotein of SEQ ID NO:62 under the control of a strong promoter sequence.Preferably, a regulatable and ubiquitous promoter sequence is used inorder to temporally control the expression of the genetic construct onceintroduced into the animal. Other methods suitable for use in thepresent methods include the use of ribozymes, antibodies, and dominantnegative forms of the present protein.

In another embodiment, the present protein provides a method to increasefibrinolysis in an individual, the method comprising administering tosaid individual an amount of the present protein sufficient to increaseplasminogen activation. The present protein is administered in any of anumber of ways, including by intravenous injection. Such methods is usedin order to eliminate clots in the prevention or the treatment ofcardiovascular diseases including, but not limited to, strokes orpulmonary embolisms.

Protein of SEQ ID NO:64 (Internal Designation Clone165843_(—)116-008-4-0-G4-F)

The cDNA of Clone 165843_(—)116-008-4-0-G4-F (SEQ ID NO:63) encodesNovel Calpastatin 1 (NC1) protein of SEQ ID NO:64, comprising the aminoacid sequence:

MTVLEITLAVILTLLGLAILAILLTRWARRKQSEMHISRYSSEQSARLLDYEDGRGSRHAYSTQSERSKRDYTPSTNSLALSRSSIALPQGSMSSIKCLQTTEELPSRTAGAMSKFFFCPLILMC FALLNC.Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:64 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 165843_(—)116-008-4-0-G4-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:64 described throughout the present application alsopertain to the nucleic acids included in Clone165843_(—)116-008-4-0-G4-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:63, 64 and Clone165843_(—)116-008-4-0-G4-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

NC1 is a physiological inhibitor of calpains. Calpains, a group ofubiquitous Ca2+-activated cytosolic proteases, have been implicated incytoskeletal remodeling events, cellular adhesion, shape change, andmobility involving site-specific regulatory proteolysis of membrane- andactin-associated cytoskeletal proteins and apoptosis [Beckerle et al.,Cell 51:569-577, 1987; Yao et al., Am. J. Physiol. 265(pt. 1):C36-46,1993; and Shuster et al., J. Cell Biol. 128:837-848, 1995; Squier etal., J. Cell Physiol., 178(3): 311-319, 1999]. Calpains have also beenimplicated in the pathophysiology of cerebral and myocardial ischemia,platelet activation, NF-kB activation, Alzheimer's disease, musculardystrophy, cataract progression and rheumatoid arthritis. There isconsiderable interest in inhibitors of calpain, as cellular adhesion,cytoskeletal remodeling events and cell mobility are linked to numerouspathologies (Wang et al., Trends in Pharm. Sci. 15:412-419, 1994; Mehdi,Trends in Biochem. Sci. 16:150-153, 1991). In addition, as thecalpain/calpastatin system is involved in membrane fusion events forseveral cell types, and calpain can be detected in human sperm andtestes extracts by Western blotting with specific antisera, tCAST maymodulate calpain in the calcium-mediated acrosome reaction that isrequired for fertilization (Li S et al., Biol Reprod, 63(1):172-8,2000).

NC1 has a unique N-terminal domain (domain L) and four repetitiveprotease-inhibitor domains (domains I-IV) (Lee W J et al., J Biol Chem,267(12):8437-42, 1992). The protein of SEQ ID NO:64 has calpastatindomains T and II. The T domain targets cytosolic localization andmembrane association, whereas domain I exhibits a nuclear localizationfunction.

NC1 plays a role in cytoskeletal remodeling events, cellular adhesion,shape change, and mobility by the site-specific regulatory proteolysisof membrane- and actin-associated cytoskeletal proteins. Preferredpolypeptides of the invention are polypeptides comprising the aminoacids of SEQ ID NO:64 from positions 1 to 116. Also preferred arefragments of SEQ ID NO:64 having a biological activity as describedtherein and the polynucleotides encoding the fragments.

One embodiment of the present invention relates to methods of using theprotein of the invention or fragment thereof in assays to detect thepresence of calpain in a biological sample, such as in bodily fluids, intissue samples, or in mammalian cell cultures. As NC1 binds calpain(Murachi, Biochemistry Int., 18(2)263-294, 1989), the protein of theinvention can be used in assays and diagnostic kits to test the presenceof calpain using techniques known to those skilled in the art.Preferably, a defined quantity of the protein of the invention orfragment thereof is added to the sample under conditions allowing theformation of a complex between the protein of the invention or fragmentthereof and heterologous proteins, and the presence of a complex and/orthe free protein of the invention or fragment thereof is assayed andcompared to a control. NC1 is useful as a marker of intracellularcalpain activation, and can be used for monitoring the involvement ofcalpain in pathological situations (De Tullio et al., FEBS letter,475(1):17-21, 2000). Calpain has been implicated in cytoskeletal proteindegradation involved in the pathophysiology of ischemia and disorderslike Alzheimer's disease (Wronski et al., J. Neural transm.,107(2):145-157, 2000) and Parkinson's disease (Mouatt-Prigent et al., J.Comp. Neurol., 419:175-92, 2000), apoptosis in neural cells of rat withspinal cord injury (SCI) (Ray, Brain res., 867(1-2):80-9, 2000), cellfusibility (Kosower et al., Methods Mol Biol., 144:181-94, 2000) andother physiopathologies. Assays detecting any increased or decreasedcalpain levels in a cell are thus useful in the diagnosis of any ofthese diseases or conditions. In addition, a recent study showed that inaddition to their proteolytic activities on cytoskeletal proteins andother cellular regulatory proteins, calpain-calpastatin systems can alsoaffect expression levels of genes encoding structural or regulatoryproteins (Chen et al., Am. J. Physiol. Cell Physiol, 279:C709-C716,2000). Thus, the ability to detect NC1 and calpain levels is also usefulfor the diagnosis of an even larger number of diseases and conditions.

In another embodiment, the polynucleotides or polypeptides of theinvention may be used for the detection of gametes, gametic precursorcells (such as spermatogenic stem cells), or of specific structureswithin the gametes, using any technique known to those skilled in theart, including those involving the use of specific antibodies andnucleic acid probes. The ability to visualize spermatozoa generally, orthe sperm acrosome in particular, has obvious utility for a number ofapplications, including for the analysis of infertility in patients.

Another embodiment of the present invention relates to a method ofinhibiting calpain in a cell, the method comprising administering to thecell an amount of the present protein sufficient to inhibit calpain inthe cell. Such methods can be performed in vitro or in vivo. Theinhibition of calpain has numerous uses in the treatment or preventionof various diseases and conditions, for example the pathophysiology ofcerebral, myocardial, renal ischemia, platelet activation, NF-kBactivation, Alzheimer's disease, Parkinson's disease, musculardystrophy, cataract progression, cancer cachexia and rheumatoidarthritis. Such an increase can be effected in any of a number of ways,including, but not limited to administering purified protein of theinvention directly to the cells, transfecting the cells with apolynucleotide encoding the protein, operably linked to a promoter; andadministering to a cell a compound that increases the activity orexpression of the protein of the invention. In addition, the expressionor activation of the protein of the invention can be inhibited in any ofa large number of ways, including using antisense oligonucleotides,antibodies, dominant negative forms of the protein, and usingheterologous compounds that decrease the expression or activation of theprotein. Such compounds can be readily identified, e.g. by screeningcandidate compounds and detecting the level of expression or activity ofthe protein using any standard assay. Other calpain inhibitors are alsoknown which can be used in conjunction with the present protein, orwhich can be used as controls in the identification of additionalinhibitors or activators of calpastatin. Such inhibitors include, butare not limited to, cerebrolysin (Wronski et al., J. Neural Transm.Suppl., 59:263-272, 2000), E-64-D (Ray et al., Brain Res., 867(1-2):80-9, 2000), and the calpain active site inhibitorN-acetyl-leucyl-leucyl-norleucinal (Squier et al., J. Cell Physiol.,178(3): 311-319, 1999).

In still another embodiment, the protein of SEQ ID:64 or fragmentthereof can be used to prevent cells from undergoing apoptosis.Specifically, any method of increasing the level or activity of thepresent protein in cells can be used to prevent the cells fromundergoing apoptosis, in vitro or in vivo. For example, a polynucleotideencoding a protein of SEQ ID NO:64, or any fragment or derivativethereof, can be introduced into cells, e.g. in a vector, wherein theprotein is expressed in the cells. Alternatively, a protein of SEQ IDNO:64 itself can be administered to cells, preferably in a formulationthat leads to the internalization of the protein by the cells. Also, anycompound that increases the expression or activation of the proteinswithin the cells can be administered. Preventing cells from undergoingapoptosis can be used for any of a large number of purposes, including,but not limited to, to prevent the death of cells being grown inculture, to prevent in a patient the apoptosis associated with any of anumber of disorders, or to prevent apoptosis in cells of a patientundergoing a treatment that increases the level of cellular stress, suchas chemotherapy. Furthermore, the invention relates to methods andcompositions using the protein of the invention or fragment thereof todiagnose, prevent and/or treat disorders characterized by abnormal cellproliferation and/or programmed cell death, including but not limited tocancer, immune deficiency syndromes (including AIDS), type I diabetes,pathogenic infections, cardiovascular and neurological injury, alopecia,aging, degenerative diseases such as Alzheimer's Disease, Parkinson'sDisease, Huntington's disease, dystonia, Leber's hereditary opticneuropathy, schizophrenia, and myodegenerative disorders such as“mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and“myoclonic epilepsy ragged red fiber syndrome” (MERRF). For diagnosticpurposes, the expression of the protein of the invention can be detectedusing any method such as Northern blotting, RT-PCR or immunoblottingmethods, and compared to the expression in control individuals, whereinan increase or decrease of the level of the present protein compared tothe control level indicates the presence of the disease or condition, orof a propensity for the disease or condition. For prevention and/ortreatment purposes of disorders in which cell proliferation needs to bereduced and/or apoptosis increased, the expression of protein of theinvention may be enhanced using any method, for example administeringthe purified protein to cells, transfecting the cells with apolynucleotide encoding the protein, or administering to the cells acompound that increases the expression or activity of the protein. Forprevention and/or treatment purposes of disorders in which cellproliferation needs to be enhanced and/or apoptosis reduced, inhibitionof endogenous expression of the protein of the invention may be achievedusing any method, including triple helix and antisense strategies.

In another embodiment, inhibiting the proteins of the invention can beused to induce apoptosis in undesired cells. Such inhibition can beaccomplished in any of a number of ways, including, but not limited to,using antibodies, antisense sequences, dominant negative forms of theprotein, or small molecule inhibitors of the expression or activity ofthe proteins. Such induction of apoptosis can be used to eliminate anyundesired cells, for example cancer cells, in a patient. Preferably,such inhibitors are targeted specifically to the undesired cells in thepatient using standard methods.

In another preferred embodiment, the protein of the invention can beused to modulate and/or characterize fertility, including for thetreatment or diagnosis of infertility, and for contraception. As NC1 isinvolved in the acrosomal reaction which is a required step infertilization, over- or under-expression or activation of the presentprotein can be used to disrupt this reaction and thereby inhibitfertility. For example, for contraception, the expression or activationof the protein can be artificially disrupted, for example by increasingthe protein level using polynucleotides encoding the protein, using theprotein itself, or using activators of protein expression or activity,or by decreasing the protein level using inhibitors such as antisenseoligonucleotides, antibodies, dominant negative forms of the protein,and using heterologous compounds that inhibit protein expression oractivity. Similarly, the cause of infertility in many patients can bedetected by detecting the level of expression of the present protein,where an abnormal level of activity or expression of the proteinindicates that a cause of infertility involves the calpain-dependentacrosomal reaction. Such a diagnosis would also point to methods oftreating the infertility, e.g. by increasing or decreasing theexpression or activation of the present protein in spermatozoa.

In another embodiment, the invention relates to methods and compositionsusing the protein of the invention or fragment thereof as a markerprotein to selectively identify tissues, preferably testis, or todistinguish between two or more possible sources of a tissue sample onthe basis of the level of the protein of SEQ ID NO:64 in the sample. Forexample, the protein of SEQ ID NO:64 or fragments thereof may be used togenerate antibodies using any techniques known to those skilled in theart, including those described therein. Such tissue-specific antibodiesmay then be used to identify tissues of unknown origin, for example,forensic samples, differentiated tumor tissue that has metastasized toforeign bodily sites, or to differentiate different tissue types in atissue cross-section using immunochemistry. In such methods a tissuesample is contacted with the antibody, which may be detectably labeled,under conditions which facilitate antibody binding. The level ofantibody binding to the test sample is measured and compared to thelevel of binding to control cells from testis or tissues other thantestis to determine whether the test sample is from testis. Similarmethods can be used to specifically detect cells expressing the protein,as well as to specifically isolate cells expressing the protein or toisolate the protein itself. For example, an antibody against the proteinof SEQ ID NO:64 or a fragment thereof may be fixed to a solid support,such as a chromatography matrix. A preparation containing cellsexpressing the protein of SEQ ID NO:64 is placed in contact with theantibody under conditions which facilitate binding to the antibody. Thesupport is washed and then the protein is released from the support bycontacting the support with agents which cause the protein to dissociatefrom the antibody.

Alternatively, the level of the protein of SEQ ID NO:64 in a test samplemay be measured by determining the level of RNA encoding the protein ofSEQ ID NO:64 in the test sample. RNA levels may be measured usingnucleic acid arrays or using techniques such as in situ hybridization,Northern blots, dot blots or other techniques familiar to those skilledin the art. If desired, an amplification reaction, such as a PCRreaction, may be performed on the nucleic acid sample prior to analysis.The level of RNA in the test sample is compared to RNA levels in controlcells from testis or tissues other than testis to determine whether thetest sample is from testis. For a number of disorders listed above,particularly of inflammatory processes, expression of the genes encodingthe polypeptide of SEQ ID NO:64 at significant higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,synovial fluid, and spinal fluid) or another tissue of cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

In another embodiment, the invention relates to methods for using theprotein of the invention or fragments to identify autoantibodies whichindicate inflammaratory processes and particularly, rheumatoid arthritis(RA), a systemic disease characterized by chronic polyarthritis andjoint destruction, and in which high levels of autoantibodies directedagainst calpastatin have been identified. Accordingly, the presentprotein may be used to detect the presence and/or the localization ofautoantibodies in a cell. In a typical embodiment, the protein of SEQ IDNO:64 is labeled with any detectable moiety including, but are notlimited to, a fluorescent label, a radioactive atom, a paramagneticion,biotin, a chemiluminescent label or a label which can be detectedthrough a secondary enzymatic or binding step. The invention furtherprovides a method of diagnosing inflammatory processes, e.g. rheumatoidarthritis, and distinguishing such processes from other diseases.

Protein of SEQ ID NO:66 (Internal Designation 335752_(—)157-154-0-B11-F)

The cDNA of Clone 335752_(—)157-15-4-0-B11-F (SEQ ID NO:65) encodesNovel Calpastatin 2 (NC2) protein of SEQ ID NO:66, comprising the aminoacid sequence:

MTVLEITLAVILTLLGLAILAILLTRWARRKQSEMYISRYSSEQSARLLDYEDGRGSRHAYSTQSERSKRDYTPSTNSLALSRSSIALPQGSMSSIKCLQTTEEPPSRTAGAMMQFTAPIPGATGPIKLSQKTIVQTLGPIVQYPGSNGRINISQLTSEDLTGAKGRVTSGPQFPNSHHVPENLHGYMNSLSLFSPA. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:66 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone335752_(—)157-15-4-0-B11-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:66described throughout the present application also pertain to the nucleicacids included in Clone 335752_(—)157-15-4-0-B11-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:65, 66 and Clone 335752_(—)157-15-4-0-B11-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

NC2 is a physiological inhibitor of calpains. Calpains, a group ofubiquitous Ca2+-activated cytosolic proteases, have been implicated incytoskeletal remodeling events, cellular adhesion, shape change, andmobility involving site-specific regulatory proteolysis of membrane- andactin-associated cytoskeletal proteins and apoptosis (Beckerle et al.,Cell 51:569-577, 1987; Yao et al., Am. J. Physiol. 265(pt. 1):C3646,1993; and Shuster et al., J. Cell Biol. 128:837-848, 1995; Squier etal., J. Cell Physiol., 178(3): 311-319, 1999). Calpains have also beenimplicated in the pathophysiology of cerebral and myocardial ischemia,platelet activation, NF-kB activation, Alzheimer's disease, musculardystrophy, cataract progression and rheumatoid arthritis. There isconsiderable interest in inhibitors of calpain, as cellular adhesion,cytoskeletal remodeling events and cell mobility are linked to numerouspathologies (Wang et al., Trends in Pharm. Sci. 15:412-419, 1994; Mehdi,Trends in Biochem. Sci. 16:150-153, 1991). In addition, as thecalpain/calpastatin system is involved in membrane fusion events forseveral cell types, and calpain can be detected in human sperm andtestes extracts.

NC2 consists of calpastatin domain T and II. The T domain targetscytosolic localization and membrane association, whereas domain I ofexhibits a nuclear localization function.

The protein of SEQ ID NO:66 is a novel member of the calpastatin familyand, as such, plays a role in cytoskeletal remodeling events, cellularadhesion, shape change, and mobility by the site-specific regulatoryproteolysis of membrane- and actin-associated cytoskeletal proteins.Preferred polypeptides of the invention are polypeptides comprising theamino acids of SEQ ID NO:66 from positions 1 to 116. Also preferred arefragments of SEQ ID NO:66 having a biological activity as describedtherein and the polynucleotides encoding the fragments.

One embodiment of the present invention relates to methods of using theprotein of the invention or fragment thereof in assays to detect thepresence of calpain in a biological sample, such as in bodily fluids, intissue samples, or in mammalian cell cultures. As NC2 binds calpain, theprotein of the invention can be used in assays and diagnostic kits totest the presence of calpain using techniques known to those skilled inthe art. Preferably, a defined quantity of the protein of the inventionor fragment thereof is added to the sample under conditions allowing theformation of a complex between the protein of the invention or fragmentthereof and heterologous proteins, and the presence of a complex and/orthe free protein of the invention or fragment thereof is assayed andcompared to a control. NC2 is useful as a marker of intracellularcalpain activation, and can be used for monitoring the involvement ofcalpain in pathological situations (De Tullio et al., FEBS letter,475(1):17-21, 2000). Calpain has been implicated in cytoskeletal proteindegradation involved in the pathophysiology of ischemia and disorderslike Alzheimer's disease (Wronski et al., J. Neural transm.,107(2):145-157, 2000) and Parkinson's disease (Mouatt-Prigent et al., J.Comp. Neurol., 419:175-92, 2000), apoptosis in neural cells of rat withspinal cord injury (SCI) (Ray, Brain res., 867(1-2):80-9, 2000), cellfusibility (Kosower et al., Methods Mol Biol., 144:181-94, 2000) andother physiopathologies. Assays detecting any increased or decreasedcalpain levels in a cell are thus useful in the diagnosis of any ofthese diseases or conditions. In addition to proteolytic activities oncytoskeletal proteins and other cellular regulatory proteins,calpain-NC2 systems can also affect expression levels of genes encodingstructural or regulatory proteins. Thus, the ability to detect NC2 andcalpain levels is also useful for the diagnosis of an even larger numberof diseases and conditions.

In another embodiment, the polynucleotides or polypeptides of theinvention may be used for the detection of gametes, gametic precursorcells (such as spermatogenic stem cells), or of specific structureswithin the gametes, using any technique known to those skilled in theart, including those involving the use of specific antibodies andnucleic acid probes. The ability to visualize spermatozoa generally, orthe sperm acrosome in particular, has obvious utility for a number ofapplications, including for the analysis of infertility in patients.

Another embodiment of the present invention relates to a method ofinhibiting calpain in a cell, the method comprising administering to thecell an amount of the present protein sufficient to inhibit calpain inthe cell. Such methods can be performed in vitro or in vivo. Theinhibition of calpain has numerous uses in the treatment or preventionof various diseases and conditions, for example, the pathophysiology ofcerebral, myocardial, renal ischemia, platelet activation, NF-kBactivation, Alzheimer's disease, Parkinson's disease, musculardystrophy, cataract progression, cancer cachexia and rheumatoidarthritis. Such an increase can be effected in any of a number of ways,including, but not limited to administering purified protein of theinvention directly to the cells, transfecting the cells with apolynucleotide encoding the protein, operably linked to a promoter; andadministering to a cell a compound that increases the activity orexpression of the protein of the invention. In addition, the expressionor activation of the protein of the invention can be inhibited in any ofa large number of ways, including using antisense oligonucleotides,antibodies, dominant negative forms of the protein, and usingheterologous compounds that decrease the expression or activation of theprotein. Such compounds can be readily identified, e.g. by screeningcandidate compounds and detecting the level of expression or activity ofthe protein using any standard assay. Other calpain inhibitors are alsoknown which can be used in conjunction with the present protein, orwhich can be used as controls in the identification of additionalinhibitors or activators of calpastatin. Such inhibitors include, butare not limited to, cerebrolysin (Wronski et al., J. Neural Transm.Suppl., 59:263-272, 2000), E-64-D (Ray et al., Brain Res., 867(1-2):80-9, 2000), and the calpain active site inhibitorN-acetyl-leucyl-leucyl-norleucinal (Squier et al., J. Cell Physiol.,178(3): 311-319, 1999).

In still another embodiment, the protein of SEQ ID:66 or fragmentthereof can be used to prevent cells from undergoing apoptosis.Specifically, any method of increasing the level or activity of thepresent protein in cells can be used to prevent the cells fromundergoing apoptosis, in vitro or in vivo. For example, a polynucleotideencoding a protein of SEQ ID NO:66, or any fragment or derivativethereof, can be introduced into cells, e.g. in a vector, wherein theprotein is expressed in the cells. Alternatively, a protein of SEQ IDNO:66 itself can be administered to cells, preferably in a formulationthat leads to the internalization of the protein by the cells. Also, anycompound that increases the expression or activation of the proteinswithin the cells can be administered. Preventing cells from undergoingapoptosis can be used for any of a large number of purposes, including,but not limited to, to prevent the death of cells being grown inculture, to prevent in a patient the apoptosis associated with any of anumber of disorders, or to prevent apoptosis in cells of a patientundergoing a treatment that increases the level of cellular stress, suchas chemotherapy. Furthermore, the invention relates to methods andcompositions using the protein of the invention or fragment thereof todiagnose, prevent and/or treat disorders characterized by abnormal cellproliferation and/or programmed cell death, including but not limited tocancer, immune deficiency syndromes (including AIDS), type I diabetes,pathogenic infections, cardiovascular and neurological injury, alopecia,aging, degenerative diseases such as Alzheimer's Disease, Parkinson'sDisease, Huntington's disease, dystonia, Leber's hereditary opticneuropathy, schizophrenia, and myodegenerative disorders such as“mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and“myoclonic epilepsy ragged red fiber syndrome” (MERRF). For diagnosticpurposes, the expression of the protein of the invention can be detectedusing any method such as Northern blotting, RT-PCR or immunoblottingmethods, and compared to the expression in control individuals, whereinan increase or decrease of the level of the present protein compared tothe control level indicates the presence of the disease or condition, orof a propensity for the disease or condition. For prevention and/ortreatment purposes of disorders in which cell proliferation needs to bereduced and/or apoptosis increased, the expression of protein of theinvention may be enhanced using any method, for example administeringthe purified protein to cells, transfecting the cells with apolynucleotide encoding the protein, or administering to the cells acompound that increases the expression or activity of the protein. Forprevention and/or treatment purposes of disorders in which cellproliferation needs to be enhanced and/or apoptosis reduced, inhibitionof endogenous expression of the protein of the invention may be achievedusing any method, including triple helix and antisense strategies.

In another embodiment, inhibiting the proteins of the invention can beused to induce apoptosis in undesired cells. Such inhibition can beaccomplished in any of a number of ways, including, but not limited to,using antibodies, antisense sequences, dominant negative forms of theprotein, or small molecule inhibitors of the expression or activity ofthe proteins. Such induction of apoptosis can be used to eliminate anyundesired cells, for example cancer cells, in a patient. Preferably,such inhibitors are targeted specifically to the undesired cells in thepatient using standard methods.

In another preferred embodiment, the protein of the invention can beused to modulate and/or characterize fertility, including for thetreatment or diagnosis of infertility, and for contraception. As NC2 isinvolved in the acrosomal reaction which is a required step infertilization, over- or under-expression or activation of the presentprotein can be used to disrupt this reaction and thereby inhibitfertility. For example, for contraception, the expression or activationof the protein can be artificially disrupted, for example by increasingthe protein level using polynucleotides encoding the protein, using theprotein itself, or using activators of protein expression or activity,or by decreasing the protein level using inhibitors such as antisenseoligonucleotides, antibodies, dominant negative forms of the protein,and using heterologous compounds that inhibit protein expression oractivity. Similarly, the cause of infertility in many patients can bedetected by detecting the level of expression of the present protein,where an abnormal level of activity or expression of the proteinindicates that a cause of infertility involves the calpain-dependentacrosomal reaction. Such a diagnosis would also point to methods oftreating the infertility, e.g. by increasing or decreasing theexpression or activation of the present protein in spermatozoa.

In another embodiment, the invention relates to methods and compositionsusing the protein of the invention or fragment thereof as a markerprotein to selectively identify tissues, preferably testis, or todistinguish between two or more possible sources of a tissue sample onthe basis of the level of the protein of SEQ ID NO:66 in the sample. Forexample, the protein of SEQ ID NO:66 or fragments thereof may be used togenerate antibodies using any techniques known to those skilled in theart, including those described therein. Such tissue-specific antibodiesmay then be used to identify tissues of unknown origin, for example,forensic samples, differentiated tumor tissue that has metastasized toforeign bodily sites, or to differentiate different tissue types in atissue cross-section using immunochemistry. In such methods a tissuesample is contacted with the antibody, which may be detectably labeled,under conditions which facilitate antibody binding. The level ofantibody binding to the test sample is measured and compared to thelevel of binding to control cells from testis or tissues other thantestis to determine whether the test sample is from testis. Similarmethods can be used to specifically detect cells expressing the protein,as well as to specifically isolate cells expressing the protein or toisolate the protein itself For example, an antibody against the proteinof SEQ ID NO:66 or a fragment thereof may be fixed to a solid support,such as a chromatography matrix. A preparation containing cellsexpressing the protein of SEQ ID NO:66 is placed in contact with theantibody under conditions which facilitate binding to the antibody. Thesupport is washed and then the protein is released from the support bycontacting the support with agents which cause the protein to dissociatefrom the antibody.

Alternatively, the level of the protein of SEQ ID NO:66 in a test samplemay be measured by determining the level of RNA encoding the protein ofSEQ ID NO:66 in the test sample. RNA levels may be measured usingnucleic acid arrays or using techniques such as in situ hybridization,Northern blots, dot blots or other techniques familiar to those skilledin the art. If desired, an amplification reaction, such as a PCRreaction, may be performed on the nucleic acid sample prior to analysis.The level of RNA in the test sample is compared to RNA levels in controlcells from testis or tissues other than testis to determine whether thetest sample is from testis. For a number of disorders listed above,particularly of inflammatory processes, expression of the genes encodingthe polypeptide of SEQ ID NO:66 at significant higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,synovial fluid, and spinal fluid) or another tissue of cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

In another embodiment, the invention relates to methods for using theprotein of the invention or fragments to identify autoantibodies whichindicate inflammatory processes and particularly, rheumatoid arthritis(RA), a systemic disease characterized by chronic polyarthritis andjoint destruction, and in which high levels of autoantibodies directedagainst calpastatin have been identified. Accordingly, the presentprotein may be used to detect the presence and/or the localization ofautoantibodies in a cell. In a typical embodiment, the protein of SEQ IDNO:66 is labeled with any detectable moiety including, but are notlimited to, a fluorescent label, a radioactive atom, a paramagnetic ion,biotin, a chemiluminescent label or a label which can be detectedthrough a secondary enzymatic or binding step. The invention furtherprovides a method of diagnosing inflammatory processes, e.g. rheumatoidarthritis, and distinguishing such processes from other diseases.

Protein of SEQ ID NO:68 (Internal Designation Clone646607_(—)181-15-2-0-E2-F)

The cDNA of Clone 646607_(—)181-15-2-0-E2-F (SEQ ID NO:67) encodesBenzodiazepine Receptor 2 (BZRP-R2) protein of SEQ ID NO:68, comprisingthe amino acid sequence:

MRLQGAIFVLLPHLGPILVWLFTRDHMSGWCEGPRMLSWCPFYKVLLLVQTAIYSVVGYASYLVWKDLGGGLGWPLALPLRLYAVQLTISWTVLVLFFTVHNPGLALLHLLLLYGLVVSTALIWHPINKLAALLLLPYLAWLTVTSALTYHLWRDSLCPVHQPQPTEKSD. Accordingly, itwill be appreciated that all characteristics and uses of thepolypeptides of SEQ ID NO:68 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 646607_(—)181-15-2-0-E2-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:68 described throughout the present application alsopertain to the nucleic acids included in Clone646607_(—)181-15-2-0-E2-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:67, 68 and Clone646607_(—)181-15-2-0-E2-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

BZRP-R2 is homologous to peripheral benzodiazepine receptor/isoquinolinebinding protein (PBR/IBP) of human, bovine and murine origin (Genbankaccession numbers M36035, M64520 and L17306 respectively). The170-amino-acid protein of SEQ ID NO:68 is similar in size andhydropathicity to known peripheral PBR/IBP benzodiazepinereceptors/isoquinoline binding proteins. BZRP-R2 has five transmembranedomains at positions 3-23,45-65, 82-102, 105-125 and 130-150. Moreover,BZRP-R2 displays a stretch of 11 amino acids (starting with V144 andending with R154) that corresponds to a recently identified putativecholesterol recognition/interaction amino acid consensus pattern(-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-) [See Li et al, Endocrinology 1998December; 139 (12): 4991-7].

BZRP-R2 is capable of binding benzodiazepine and imidazopyridinederivatives, but is distinct from the GABA neurotransmitter receptor.BZRP-R2 polypeptides are most abundant in steroidogenic cells and arefound primarily on outer mitochondrial membranes. BZRP-R2 is associatedwith a 34-kDa pore-forming, voltage-dependent anion channel proteinlocated on the outer/inner mitochondrial membrane contact sites. Ligandsof BZRP-R2, upon binding to the receptor, simulate steroid synthesis insteroidogenic cells in vitro and in vivo. BZRP-R2 stimulates steroidformation by increasing the rate of cholesterol transfer from the outerto the inner mitochondrial membrane.

In addition to its role in mediating cholesterol movement acrossmembranes, BZRP-R2 has been implicated in several other physiologicalfunctions, including cell growth and differentiation, chemotaxis,mitochondrial physiology, porphyrin and heme biosynthesis, immuneresponse, and anion transport. In addition, BZRP-R2 agonists are potentanti-apoptotic compounds.

BZRP-R2 is associated with stress and anxiety disorders. BZRP-R2 plays arole in the regulation of several stress systems such as the HPA axis,the sympathetic nervous system, the renin-angiotensin axis, and theneuroendocrine axis. In these systems, acute stress typically leads toincreases in BZRP-R2 density, whereas chronic stress typically leads todecreases in BZRP-R2 density. For example, in Generalized AnxietyDisorder (GAD), Panic Disorder (PD), Generalized Social Phobia (GSP),and Post-Traumatic Stress Disorders (PTSD), BZRP-R2 density is typicallydecreased. BZRP-R2 is expressed glial cells in the brain. Furthermore,BZRP-R2 expression is increased in neurodegenerative disorders and afterneurotoxic and traumatic-ischemic brain damage. BZRP-R2 expression isdecreased in chronic schizophrenics, suggesting that the decreaseddensity of BZRP-R2 in the brain may be involved in the pathophysiologyof schizophrenia. However, BZRP-R2 is higher than normal in autopsiedbrain tissue from PSE patients (Portal-Systemic Encephalopathypatients).

BZRP-R2 increases mitochondrial activity and prevents apoptosis and istherefore implicated tumor cell proliferation. BZRP-R2 is preferentiallyexpressed in liver and breast cancers. Further, BZRP-R2 is useful as atool/marker for detection, diagnosis, prognosis and treatment of cancer.

Many ligands have been described that bind to BZRP-R2 with variousaffinities. Some benzodiazepines, Ro 5-4864 [4-chlorodiazepam], diazepamand structurally related compounds, are potent and selective PBRligands. Exogenous ligands also include 2-phenylquinoline carboxamides(PK11195 series), imidazo [1,2-a]pyridine-3-acetamides (Alpidem series),pyridazine, and isoquinilone derivatives. Some endogenous compounds,including porphyrins and diazepam binding inhibitor (DBI), bind toBZRP-R2.

In one embodiment, a preferred polypeptide of the invention comprisesthe amino acids of SEQ ID NO:68 from position 144 to 154: VTSALTYHLWR.Further preferred fragments of BZRP-R2 comprise the epitope: ALPLRLYAVor fragments thereof. In another embodiment, the subject inventionprovides a polypeptide comprising the sequence of SEQ ID NO:68. Otherpreferred polypeptides of the invention include biologically activefragments of SEQ ID NO:68. Biologically active fragments of the proteinof BZRP-R2 have any of the biological activities described herein. Inanother embodiment, the polypeptide of the invention is encoded by clone646607 215-15-5-0-B11-F.

A preferred embodiment of the invention is a method of screening forcompounds that modulate the expression of BZRP-R2. This method comprisesthe steps of i) contacting a cell with a test compound and ii) comparingthe level of BZRP-R2 polypeptides in a cell after exposure to the testcompound to that of an untreated control cell. The level of BZRP-R2polypeptides may be inferred by detecting mRNA for BZRP-R2 by methodscommon to the art such as Northern blotting or RT-PCR. The level ofBZRP-R2 polypeptides may also be detected by antibody-based methodscommon to the art such as Western blotting or immunofluorescence. Testcompounds that increase BZRP-R2 expression are useful as agonists, asdiscussed herein. Test compounds that decrease BZRP-R2 expression areuseful as antagonists, as discussed herein.

Antagonists of BZRP-R2 include agents which decrease the levels ofexpressed mRNA encoding the protein of SEQ ID NO:68. These include, butare not limited to, RNAi, one or more ribozymes capable of digesting theprotein of the invention, or antisense oligonucleotides capable ofhybridizing to mRNA encoding BZRP-R2. Antisense oligonucleotides can beadministrated as DNA, RNA, as DNA entrapped in proteoliposomescontaining viral envelope receptor proteins [Kanoda, Y. et al. (1989)Science 243: 375, which disclosure is hereby incorporated by referencein its entirety] or as part of a vector which can be expressed in thetarget cell to provide antisense DNA or RNA. Vectors which are expressedin particular cell types are known in the art. Alternatively, the DNAcan be injected along with a carrier. A carrier can be a protein such asa cytokine, for example interleukin 2, or polylysine-glycoproteincarriers. Carrier proteins, vectors, and methods of making and usingpolylysine carrier systems are known in the art. Alternatively, nucleicacid encoding antisense molecules may be coated onto gold beads andintroduced into the skin with, for example, a gene gun [Ulmer, J. B. etal. (1993) Science 259:1745, which disclosure is hereby incorporated byreference in its entirety].

A preferred embodiment of the invention is a method of screening forcompounds that bind to BZRP-R2 polypeptides. Such compounds are usefulfor developing agonists and antagonists of BZRP-R2 activity. This methodcomprises the steps of: i) contacting a BZRP-R2 polypeptide or fragmentthereof with a test compound under conditions that allow binding tooccur and ii) detecting binding of said test compound. Binding may bedetected by any method common to the art such as competition with alabeled antibody specific for BZRP-R2 or by direct labeling of each testsubstance. In one example of such a method, a polynucleotide encoding aBZRP-R2 polypeptide or a biologically active fragment thereof istransformed into a eukaryotic or prokaryotic host cell. The transformedcells may be viable or fixed. Drugs or compounds which are candidatesfor binding BZRP-R2 polypeptides are screened against such transformedcells in binding assays well known to those skilled in the art.Alternatively, assays such as those taught in Geysen H. N., WOApplication 84/03564, published on Sep. 13, 1984, and incorporatedherein by reference in its entirety, may be used to screen for peptidecompounds which demonstrate binding affinity for BZRP-R2 polypeptides orfragments thereof. In another embodiment, competitive drug screeningassays using neutralizing antibodies specifically compete with a testcompound for binding to BZRP-R2 polypeptides or fragments thereofPreferred test compounds are those included in the benzodiazepine class,such as diazepam (i.e., valium), triazolobenzodiazepine, and adinazolam,as well as modified versions thereof. Further preferred test compoundsare in the imidazo pyridine and isoquinilone classes.

A variety of drug screening techniques may be employed. In this aspectof the invention, BZRP-R2 polypeptide or biologically active fragmentsthereof, may be free in solution, affixed to a solid support,recombinantly expressed on or chemically attached to a cell surface, orlocated intracellularly. The formation of binding complexes betweenBZRP-R2 polypepetides or biologically active fragments thereof, and thecompound being tested, may then be measured as described.

Another embodiment of the subject invention provides compositions andmethods of selectively modulating the activity of the protein of theinvention. Modulation of BZRP-R2 allows for the successful prevention,treatment, or management of disorders or biochemical abnormalitiesassociated with BZRP-R2. Agonist compounds are those that increase theamount of BZRP-R2 polypeptides in a cell or increase the biologicalactivity of BZRP-R2. A preferred embodiment of the invention is a methodof screening for agonists that bind to BZRP-R2 comprising the steps of:i) screening for test substances that bind to BZRP-R2, as describedabove and ii) detecting BZRP-R2 biological activity. Preferably, thismethod is accomplished in an intact cell. Further preferably, the cellis a steroidogenic cell such as a testicular or ovarian cell.Preferably, the biological activity of BZRP-R2 is determined bymeasuring the concentration of steroid hormones released from the cellbefore and after exposure to the test substance. Agonists of BZRP-R2will increase the release of steroid hormones from the cell. Antagonistcompounds are those that decrease the amount of BZRP-R2 polypeptides ina cell or decrease the biological activity of BZRP-R2. Another preferredembodiment of the invention is a method of screening for antagoniststhat bind to BZRP-R2 comprising the steps of: i) screening for testsubstances that bind to BZRP-R2, as described above and ii) detectingBZRP-R2 biological activity. Preferably, this method is accomplished inan intact cell. Further preferably, the cell is a steroidogenic cellsuch as a testicular or ovarian cell. Preferably, the biologicalactivity of BZRP-R2 is determined by measuring the concentration ofsteroid hormones released from the cell before and after exposure to thetest substance. Antagonists of BZRP-R2 will decrease the release ofsteroid hormones from the cell.

Antagonists, able to reduce or inhibit the expression or the activity ofthe protein of the invention, are useful in the treatment of diseasesassociated with elevated levels of BZRP-R2, increased cell proliferationor reduced apoptosis, and increased cholesterol transport. Thus, thesubject invention provides methods for treating a variety of diseases ordisorders, including but not limited to cancers, especially liver andbreast cancer, and portal-systemic encephalopathy. Increased cholesteroltransport into the mitochondria of steroidogenic cells results in higherthan normal production of steroid hormones such as progesterone,testosterone, and estrogen. Abnormally high levels of steroid hormoneslead to disruption of adrenocortical feedback mechanisms andunderproduction of trophic hormones from the hypothalamus and pituitary.Inhibition of BZRP-R2 and steroidogenesis may increase levels of trophichormones such as gonadotropin-releasing hormone.

Alternatively, the subject invention provides a method of treatingdiseases or disorders associated with decreased levels of BZRP-R2polypeptides and decreased steroid hormone release with an agonistthereof. Such method comprises the step of contacting a cell with aBZRP-R2 agonist. This method comprises the step of contacting a cellwith an agonist of BZRP-R2. Thus, the subject invention provides methodsof treating disorders including, but not limited to, schizophrenia,chronic stress, GAD, PD, GSP and PTSD. Other disorders which may betreated by agonists of BZRP-R2 include those associated with decreasesin cell proliferation, e.g. developmental retardation. Furthermore,because BZRP-R2 is able to transport cholesterol into cells, BZRP-R2agonists may also be used to increase cholesterol transport into cells.Diseases associated with cholesterol transport deficiencies includelipoidal adrenal hyperplasia, ovarian cysts, abnormal lipid deposits insteroidogenic cells. Disorders that reflect a requirement forcholesterol for myelin and myelination, include Alzheimer's disease,multiple sclerosis, spinal cord injury, and brain developmentneuropathy. The methods of treating disorders associated with decreasedlevels of BZRP-R2 may be practiced by introducing agonists whichstimulate the expression or the activity of BZRP-R2.

Additionally, disorders resulting from defective mitochondrial activitymay be treated with an agonist to BZRP-R2. Defective mitochondrialactivity may alternatively or additionally result in the generation ofhighly reactive free radicals that have the potential of damaging cellsand tissues. These free radicals may include reactive oxygen species(ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and otherreactive species that may be toxic to cells and cause apoptosis. Forexample, oxygen free radical induced lipid peroxidation is awell-established pathogenic mechanism in central nervous system (CNS)injury such as that found in a number of degenerative diseases, and inischemia (i.e., stroke). Diseases associated with altered mitochondrialfunction and apoptosis include: Alzheimer's Disease, diabetes mellitus,Parkinson's Disease, Huntington's disease, dystonia, Leber's hereditaryoptic neuropathy, schizophrenia, mitochondrial encephalopathy, lacticacidosis, and stroke.

A further preferred embodiment includes a method of inhibiting apoptosisof cells in culture. This method comprises the step of contacting a cellin culture with an agonist to BZRP-R2. Such methods are useful forculturing cells that are notoriously undergo apoptosis, such as primaryneurons and lymphocytes.

In one embodiment, the level of BZRP-R2 in a cell may be increased byintroducing nucleic acids encoding a BZRP-R2 polypeptide or biologicallyactive fragment thereof into a targeted cell type. Vectors useful insuch methods are known to those skilled in the art, as are methods ofintroducing such nucleic acids into target tissues.

Antibodies or other polypeptides capable of reducing or inhibiting theactivity of BZRP-R2 may be provided as in isolated and substantiallypurified form. Alternatively, antibodies or other polypeptides capableof inhibiting or reducing the activity of BZRP-R2 may be recombinantlyexpressed in the target cell to provide a modulating effect. Inaddition, compounds which inhibit or reduce the activity of BZRP-R2 maybe incorporated into biodegradable polymers being implanted in thevicinity of where drug delivery is desired. For example, biodegradablepolymers may be implanted at the site of a tumor or, alternatively,biodegradable polymers containing antagonists/agonists may be implantedto slowly release the compounds systemically. Biodegradable polymers,and their use, are known to those of skill in the art (see, for example,Brem et al. (1991) J. Neurosurg. 74:441-446, which disclosure is herebyincorporated by reference in its entirety).

In another embodiment, the invention provides methods and compositionsfor detecting the level of expression of the mRNA encoding the proteinof the invention. Quantification of mRNA levels of BZRP-R2 may be usefulfor the diagnosis or prognosis of diseases associated with an alteredexpression of the protein of the invention. Assays for the detection andquantification of the mRNA encoding BZRP-R2 are well known in the art(see, for example, Maniatis, Fitsch and Sambrook, Molecular Cloning; ALaboratory Manual (1982), or Current Protocols in Molecular Biology,Ausubel, F. M. et al. (Eds), Wiley & Sons, Inc., disclosures of whichare hereby incorporated by reference in their entireties).

Polynucleotides probes or primers for the detection of BZRP-R2 nRNA canbe designed from the cDNA of SEQ ID NO:67. Methods for designing probesand primers are known in the art. In another embodiment, the subjectinvention provides diagnostic kits for the detection of the mRNA of theprotein of the invention in cells. The kit comprises a package havingone or more containers of oligonucleotide primers for detection of theprotein of the invention in PCR assays or one or more containers ofpolynucleotide probes for the detection of the mRNA of the protein ofthe invention by in situ hybridization or Northern analysis. Kits may,optionally, include containers of various reagents used in varioushybridization assays. The kit may also, optionally, contain one or moreof the following items: polymerization enzymes, buffers, instructions,controls, or detection labels. Kits may also, optionally, includecontainers of reagents mixed together in suitable proportions forperforming the hybridization assay methods in accordance with theinvention. Reagent containers preferably contain reagents in unitquantities that obviate measuring steps when performing the subjectmethods.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with altered levels ofthe protein of the invention. Diagnostic assays to detect the protein ofthe invention may comprise a biopsy, in situ assay of cells from organor tissue sections, or an aspirate of cells from a tumor or normaltissue. In addition, assays may be conducted upon cellular extracts fromorgans, tissues, cells, urine, or serum or blood or any other body fluidor extract.

Assays for the quantification of BZRP-R2 polypeptides may be performedaccording to methods well known in the art. Typically, these assayscomprise the steps of: contacting the sample with a ligand of theprotein of the invention or an antibody (polyclonal or monoclonal) thatspecifically recognizes the protein of the invention or a fragmentthereof and detecting the complex formed between the protein of theinvention present in the sample and the ligand or antibody. Fragments ofthe ligands and antibodies may also be used in the binding assays,provided these fragments are capable of specifically interacting withBZRP-R2 polypeptides. Further, ligands and antibodies which bind toBZRP-R2 may be labeled according to methods known in the art. Labelswhich are useful in the subject invention include, but are not limitedto, enzymes labels, radioisotopic labels, paramagnetic labels, andchemiluminescent labels. Typical techniques are described by Kennedy, J.H., et al. (1976) Clin. Chim. Acta 70:1-31; and Schurs, A. H. et al.(1977) Clin. Chim. Acta 81: 1-40, disclosures of which are herebyincorporated by reference in their entireties.

The subject invention also provides methods and compositions for theidentification of metastatic tumor masses. In this aspect of theinvention, the polypeptide or antibody that specifically binds a BZRP-R2polypeptide or fragment thereof may be used as a marker for theidentification of the metastatic tumor mass. Metastatic tumors whichoriginated from the breast or liver may overexpress BZRP-R2polypeptides, whereas newly forming tumors, or those originating fromother tissues are not expected to bear BZRP-R2.

Protein of SEQ ID NO:70 (Internal Designation Clone229654_(—)114-049-1-0-F12-F (cFS))

The cDNA of Clone 229654_(—)114-049-1-0-F12-F (SEQ ID NO:69) encodes the787 amino acid long polypeptide called LAP of SEQ ID NO:70 comprisingthe amino acid sequence:

MFRLWLLLAGLCGLLASRPGFQNSLLQIVIPEKIQTNTNDSSEIEYEQISYIIPIDEKLYTVHLKQRYFLTDNFMIYLYNQGSMNTYSSDIQTQCYYQGNIEEYPDSMVTLSTCSGLRGILQFENVSYGIEPLESAVEFQHVLHKLKNEDNDIAIFIDRSLKEQPMDDNIFISEKSEPAVPDLFPLYLEMHIVVDKTLYDYWGSDSMIVTNKVIEIVGLANSMFTQFKVTIVLSSLELWSDENKISTVGEADELLQKFLEWKQSYLNLRPHDIAYLLIYMDYPRYLGAVFPGTMCITRYSAGVALYPKEITLEAFAVIVTQMLALSLGISYDDPKKCQCSESTCIMNPEVVQSNGVKTFSSCSLRSFQNFISNVGVKCLQNKPQMQKKSPKPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECRPKAHPECDIAENCNGSSPECGPDITLINGLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVIYAFVRDSVCITVDYKLPRTVPDPLAVKNGSQCDIGRVCVNRECVESRIIKASAHVCSQQCSGHGVCDSRNKCHCSPGYKPPNCQIRSKGFSIFPEEDMGSIMERASGKTENTWLLGFLIALPILIVTTAIVLARKQLKNWFAKEEEFPSSESKSEGSTQTYASQSSSEGSTQTYAGQTRSESSSQADTSKSKSEDSAEAYTSRSKSQDSTQTQSSSN. Accordingly, it willbe appreciated that all characteristics and uses of polypeptides of SEQID NO:70 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone229654_(—)114-049-1-0-F12-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:69described throughout the present application also pertain to the nucleicacids included in Clone 229654_(—)114-049-1-0-F12-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:69, SEQ ID NO:70, and Clone 229654_(—)114-049-1-0-F12-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

LAP, the protein of SEQ ID NO:70, is a new member of the ADAM (ADisintegrin And Metalloprotease domain) family of proteins. The gene forClone 229654.cFS is located on chromosome 8 and is expressed in tissuesincluding liver, adipose and testis.

LAP, as an ADAM family member is a membrane-anchored cell surfaceprotein. The members of this family form a large group of cell surfaceadhesion molecules and proteases whose name describes the two domainsthat these proteins share with their closest relatives, the PIII classof snake venom metalloproteinases (SVMPs). The ADAM proteases fall withthe SVMPS within the adamalysin/reprolysin subfamily of Zinc-dependentmetalloproteinases. ADAMs have also been referred to as MDCs(metalloproteinase/disintegrin/cysteine-rich), cellular disintegrins,and metalloproteinase-desintegrins.

These proteins have been isolated from a wide range of organisms rangingfrom yeast, worm, flies, frogs and mammals. Expression studies haveshown that while some ADAMS have wide tissue expression, some have theirexpression restricted to one tissue.

The ADAM proteins have been shown to function in cell-cell interaction,cell-signaling, and in the processing of the ectodomains ofmembrane-anchored proteins and have been implicated in diversebiological processes, including sperm-egg binding and fusion, myoblastfusion, protein-ectodomain shedding of cytokines, cytokine receptors,adhesions and other extracellular protein domains. Furthermore, theyhave been shown to be necessary for proper axonal guidance, neural andwing development in Drosophila, vulval development in Caenorhabditiselegans, and epithelial maturation and skin and hair development in themouse.

Structurally, LAP has an N-terminal signal sequence (MFRLWLLLAGLCGLLAS),a prodomain

(HLKQRYFLTDNFMIYLYNQGSMNTYSSDIQTQCYYQGNIEEYPDSMVTLSTCSGLRGILQFENVSYGIEPLESAVEFQHVLHKLKNEDNDIAIFIDRSLKEQPMDDNIFISEKS) that has beenshown to maintain the enzyme in an inactive state, followed by ametalloprotease domain

(LYLEMHIVVDKTLYDYWGSDSMIVTNKVIEIVGLANSMFTQFKVTIVLSSLELWSDENKISTVGEADELLQKFLEWKQSYLNLRPHDIAYLLIYMDYPRYLGAVFPGTMCITRYSAGVALYPKEITLEAFAVIVTQMLALSLGISYDDPKKCQCSESTCIMNPEVVQSNGVKTFSSCSLRSFQNFISNVGVKCLQNKP) that is important for proteolysis and contains thezinc-binding catalytic site, a disintegrin-like domain(KPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECPKAHPECDIAENCNGS SPEC) that has been demonstrated to bind integrins, acystein-rich region(GLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVIYAFVRDSVC) that also have adhesionactivity, an EGF-like domain(CDIGRVCVNRECVESRIIKASAHVCSQQCSGHGVCDSRNKCHCSPGYKPPNC) important forsubstrate recognition, a transmembrane domain (TWLLGFLIALPILIVTTAIVL)and a cytoplasmic tail(ARKQLKNWFAKEEEFPSSESKSEGSTQTYASQSSSEGSTQTYAGQTRSESSSQADTSKSKSEDSAEAYTSRSKSQDSTQTQSSSN) that has been shown in many ADAMs to containSH3 binding sites and which might be important for cell signaling.

Interestingly, the cytoplasmic C-terminal domain of the LAP protein doesnot contains any SH3 binding sites but it ends by a 69 amino acid regionrich in serine/threonine residues (36% of serine residues;

SSESKSEGSTQTYASQSSSEGSTQTYAGQTRSESSSQADTSKSKSEDSAEAYTSRSKSQDST QTQSSS).

LAP contains both a disintegrin-like and a metalloprotease domain, andhas both cell adhesion and protease activities. However, LAP lacks thecatalytic site consensus sequence in its metalloprotease domain(QMLALSLGISYD). LAP, like fertilin beta another catalytically inactiveprotease, is processed on the sperm cell surface during sperm maturationin the epididymus yielding mature protein that retains disintegrindomain on fertilization-competent sperm.

Preferred LAP polypeptides for uses in the methods described belowinclude the polypeptides comprising the amino sequence of:

KPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECRPKAHPECDIAENCNGSSPECGPDITLINGLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVIYAFVRDSVCITVDYKLPRTVPDPLAVKNGSQCDIGRVCVNRECVESRIIKASAHVCSQQCSGHGVCDSRNKCHCSPGYKPPNCQIRSKGFSIFPEEDMGSIMERASGKTENTWLLGFLIALPILIVTTAIVLARKQLKNWFAKEEEFPSSESKSEGSTQTYASQSSSEGSTQTYAGQTRSESSSQADTSKSKSEDSAEAYTSRSKSQDSTQTQSSSN;

A polypeptide comprising the amino acid sequence of:

KPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECRPKAHPECDIAENCNGSSPECGPDITLINGLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVIYAFVRDSVCITVDYKLPRTVPDPLAVKNGSQCDIGRVCVNRECVESRIIKASAHVCSQQCSGHGVCDSRNKCHCSPGYKPPNCQIRSKGFSIFPEEDMGSIMERASGKTEN

A polypeptide comprising the amino acid sequence of:

KPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECRPKAHPECDIAENCNGSSPECGPDITLINGLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVI YAFVRDSVC

A polypeptide comprising the amino acid sequence of:

KPVCGNGRLEGNEICDCGTEAQCGPASCCDFRTCVLKDGAKCYKGLCCKDCQILQSGVECRPKAHPECDIAENCNGSSPECGPD

A polypeptide comprising the amino acid sequence of:

GLSCKNNKFICYDGDCHDLDARCESVFGKGSRNAPFACYEEIQSQSDRFGNCGRDRNNKYVFCGWRNLICGRLVCTYPTRKPFHQENGDVIYAFVRDSVC

A polypeptide comprising the amino acid sequence of:

PSSESKSEGSTQTYASQSSSEGSTQTYAGQTRSESSSQADTSKSKSEDSAEAYTSRSKSQDS TQTQSSSN

An embodiment of the invention is directed to a method to screen formolecules which block the interaction of LAP with the cell-surfacereceptors on the oocyte surface comprising the steps of contacting spermwith said molecule to be screen, contacting the sperm with the oocyte,and disrupting sperm-oocyte binding.

A preferred embodiment of the invention is directed to a method ofinhibiting sperm-oocyte interaction by blocking the LAP interaction withthe oocyte cell surface comprising the steps of contacting sperm with ablocking molecule, as identified in a screen, which inhibits or blocksthe LAP-oocyte interaction. Preferred agents include antibodies directedto LAP-disintegrin domain.

LAP is a plasma membrane-anchored protein having adhesion and cellsignaling activities in liver cells as well as in adipocytes. Morespecifically, it is believed that the mature LAP protein interacts, viaits extracellular domain, with as yet unidentified integrins and otherproteins present at the surface of neighbouring cells, while itscytoplasmic serine-rich domain is involved in signaling events byinteracting with cytoplasmic or plasma membrane-associated proteins thatinteract with serine-rich domains. More over, as serine and threonineresidues are both phosphorylatable residues, the signaling activity ofthe LAP protein is regulated by phosphorylation/dephosphorylation eventsof specific serine and or threonine residue(s) present on this domain.

In a further embodiment, polyclonal or monoclonal antibodies directedagainst polypeptides of the invention are used in methods to reduce orinhibit cell-cell interactions between cells in vitro, preferably liveror adipose cells. A preferred method of reducing or inhibiting cell-cellinteractions comprises the steps: i) contacting the cells with acomposition comprising an inhibitory-effective amount of an antibodydirected against polypeptides of the invention, preferably a monoclonalantibody directed against the disintegrin-like domain or a monoclonalantibody directed against the cysteine-rich domain.

A further embodiment is directed to a method of blocking or inhibitingthe interaction of LAP with at least one of its binding-partnerscomprising the steps: i) contacting cells with a blocking-effectiveamount of a polypeptide fragment of the invention comprising anextracellular domain of LAP. Preferred extracellular domains to be usedin said methods of blocking the interaction of LAP and a binding partnerinclude the disintegrin-like domain of LAP and the cysteine-rich domainof LAP. Preferred synthetic peptides to be used in compositions of saidmethods have amino acid sequences comprising CRPKAHPECDIAENC orCGNGRLEGNEICDCG, or a combination thereof.

Protein of SEQ ID:72 (Internal Designation Clone338116_(—)174-1-1-0-B10-F)

The cDNA of Clone 338116_(—)174-1-1-0-B10-F (SEQ ID NO:71) encodes theprotein of SEQ ID NO:72, herein referred as Short Histone Deacetylase(SHDAC), comprising the amino acid sequence:

MGPHLHLCLCVPDLRSLRVCVSLWSVHHRPHESLAREEALTALGKLLYLLDGMLDGQVNSGIAATPASAAAATLDVAVRRGLSHAAQRLLCVALGQLDRPPDLAHDGRSLWLNIRGKEAAALSMFHVSTPLPVMTGGFLSCILGLVLPLAYGFQPDLVLVALGPGHGLQGPHXALLAAMLRGLAGGRVLALLEENSTPQLAGILARVLNGEAPPSLGPSSVASPEDVQALMYLRGQLEPQWKMLQCHPHLVA, is encoded by the cDNA clone 338116_(—)174-1-1-0-B10-F(SEQ ID:71). The protein of SEQ ID NO:72 is a novel variant of histonedeacetylase (HDAC). Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptide of SEQ ID NO:72 describedthroughout the present application also pertain to the polypeptideencoded by a nucleic acid included in clone 338116_(—)174-1-1-0-B10-F.In addition, it will be appreciated that all characteristics and uses ofthe nucleic acid of SEQ ID NO:71 described throughout the presentapplication also pertain to the nucleic acid included in clone338116_(—)174-1-1-0-B10-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:71, SEQ ID NO:72, andClone 338116_(—)174-1-1-0-B10-F. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding the fragments.

The protein of SEQ ID:72 contains one potential transmembrane segment(position 130 to 150), and a signal peptide (position 1:MGPHLHLCLCVPDLRSL). The protein of SEQ ID:72 is highly expressed inplacenta and salivary glands.

Histone deacetylase (HDAC) proteins comprise a family of relatedproteins that act in conjunction with histone acetyl-transferaseproteins to modulate chromatin structure and transcriptional activityvia changes in the acetylation status of histones. HDACs remove acetylgroups from histones by hydrolysis [Davie, J. R. Curr. Opin. Genet. Dev.8, 173-178 (1998)], thereby causing local chromatin condensation anddecreasing the accessibility of particular DNA regions for RNApolymerase complexes. In fact, transcriptionally active chromatincorrelates with histone hyperacetylation [Grundstein M. Nature389:349-352 (1997)], and it has been suggested that histoneacetyltransferases promote transcription while histone deacetylases actas repressors and transcriptional silencer [Doetzlhofer A. et al., Mol.Cell. Biol. 19:5504-5511(1999)].

Histone deacetylase proteins belong to a superfamily of zincmetalloenzymes with a conserved 380 residue catalytic domain [Finnin, M.S. et al., Nature 401:188-193(1999)]. Histone deacetylases are found inhigh-molecular-weight complexes associated with adapter proteins likeSIN3, RbAp46/48, SAP18, SAP30, and nuclear corepressors like N-CoR,SMRT, and SUN-CoR [Alland, L. et al., Nature 387:49-55 (1997); Heinzel,T. et al., Nature 387:43-48 (1997); Laherty, C. D. et al., Cell 89:349-356 (1997); Nagy, L. H. et al., Cell 89:373-380 (1997); Zhang, W. etal., EMBO J. 17:3155-3167 (1997); Zhang; Y. et al., Cell 89:357-364(1997); Knoepfker, P. S. & Eisenman, R. N. Cell 99:447-450 (1999)].

Histone deacetylases are recruited to specific promoters by mammaliantranscriptions factors such as Matrix-associated Deacetylase (Mad)[Sommer, A. et al., Curr. Biol. 7:357-365 (1997)], YY1 [Yang, W. M.Proc. Natl. Acad. Sci. USA 93:12845-12850 (1996)], hormone-dependentnuclear receptor [Nagy, L. H. et al., Cell 89:373-380 (1997)], MeCP2[Jones, P. L. et al., Nat. Genet. 19:187-191 (1998)], CBF [Kao, H. Y. P.et al., Genes Dev. 12:2269-2277 (1998)], Retinoblastoma protein (Rb)[Brehm, A. et al., Nature 391:597-601 (1998)], groucho [Chen, G. et al.,Genes Dev. 13:2218-2230 (1999)] B-lymphocyte-induced maturation protein[Yu, J. et al., Mol. Cell. Biol. 20:2592-2603 (2000)] and related pocketproteins [Ferreira et al., Proc. Natl. Acad. Sci. USA 95:10493-10498(1998)] for repression. The recruitment of human histone deacetylases byPZLF (promyelocytic leukaemia zinc finger), PML promyelocyticleukaemia), and ETO fusion proteins can interfere with differentiationof hematopoietic precursor cells in acute promyelocytic leukemia [Lin,R. J. et al., Nature 311:811-815 (1996); David, G. L. et al., Oncogene16:25492556 (1998); Grignani, F. S. et al., Nature 391:815-818 (1998);Guidez et al., Blood 91:2634-2642 (1998)].

Several drugs have been identified as acting upon histone acetylation.Some examples are: trichostatin A (TSA), apicidin (antiprotozoal agent),superoylanilide hydroxamic acid (SAHA), cyclic hydroxamicacid-containing peptide (CHAP) 1, FR901228 (a potent antitumor), CBHA(m-carboxycinnamic acid bis-hydroxamide), trapoxin, MS-275 (antitumor),pyroxamide (suberoyl-3-aminopyridineamide hydroxamic) acid and phenylbutyrate. Such drugs cause major alterations in cellular activity,including the induction of cellular differentiation and apoptosis[Medina, V. et al., Cancer Res. 57:3697-3707 (1997); Richon, V. M. etal., Proc. Natl. Acad. Sci. U.S.A. 95:3003-3007 (1998); Sambucetti, L.C. et al., J. Biol. Chem. 274:34940-34947 (1999); Buter, L. M. et al.,Clin. Cancer Res. 7:962-970 (2001); Coffey, D. C. et al., Cancer Res.61:3591-3594 (2001); Colletti, S. L. et al., Bioorg. Med. Chem. Lett.11:107-111 (2001); Furumai, R. et al., Proc. Natl. Acad. Sci. USA98:87-92. (2001); Lee, B. I. et al., Cancer Res. 63:931-934 (2001)].

The protein of SEQ ID NO:72 is a novel splice and polymorphism variantof histone deacetylase and, as such, plays a role in transcription,chromosome stability, cell cycle progression, gene silencing, lymphocyteand muscle differentiation, aging, regulation of neuronal phenotype, DNAreplication and the response to DNA damage. Particularly, the protein ofthe invention may deacetylate substrates, preferably acetylatedhistones, either directly or indirectly as enzymes cofactors. Preferredpolypeptides of the invention are polypeptides comprising the aminoacids of SEQ ID NO:72 from positions 29 to 252. Also preferred arefragments of SEQ ID NO:72 having a biological activity as describedtherein and the polynucleotides encoding the fragments. Thedeacetylation activity of the protein of the invention or fragmentthereof may be assayed using any of a number of methods known to thoseskilled in the art.

The invention relates to methods and compositions using the protein ofSEQ ID NO:72 or fragment thereof to inhibit or modulate cellulartranscriptional activity, thereby modulating cellular differentiation.Specifically, as histone deacetylases play a role in inhibitingtranscription associated with differentiation, then an increase in theactivity or expression of the protein can be used to inhibitdifferentiation. The ability to inhibit differentiation has a number ofuses, for example during the cultivation of undifferentiated pluripotentcells to maintain the cultured cells in an undifferentiated state untilthe need for a given cell type arises (in cases of grafts for instance).For example, the histone deacetylase of the invention may be used toarrest a population of non-neoplastic cells grown in vitro in the G1 orG2 phase of the cell cycle. Such synchronization allows, for example,the identification of gene and/or gene products expressed during the G1or G2 phase of the cell cycle. Such a synchronization of cultured cellsmay also be useful, for testing the efficacy of a new transfectionprotocol, where transfection efficiency varies and is dependent upon theparticular cell cycle phase of the cell to be transfected. Use of thehistone deacetylase of the invention allows the synchronization of apopulation of cells, thereby adding detection of enhanced transfectionefficiency. The level of the protein activity or expression can beincreased in any of a number of ways, including by introducing apolynucleotide encoding the protein into cells, by administering theprotein itself to cells, or by administering to cells a compound thatincreases protein activity or expression. Alternatively, the expressionor activation of the protein of the invention can be inhibited in any ofa large number of ways, including using antisense oligonucleotides,antibodies, dominant negative forms of the protein, and usingheterologous compounds that decrease the expression or activation of theprotein. Such compounds can be readily identified, e.g. by screeningcandidate compounds and detecting the level of expression or activity ofthe protein using any standard assay. The ability to promotedifferentiation has many uses, including in the treatment or preventionof cancer, as cancer cells are often in a relatively undifferentiatedstate, and cellular differentiation typically accompanies by growtharrest.

In another embodiment, eukaryotic cells are genetically engineered inorder to express the protein of the invention or fragment thereof underspecific conditions in order to prevent and/or treat disorderscharacterized by abnormal cell proliferation and/or programmed celldeath, including but not limited to cancer, immune deficiency syndromes(including AIDS), type I diabetes, pathogenic infections, cardiovascularand neurological injury, alopecia, aging, degenerative diseases such asAlzheimer's Disease, Parkinson's Disease, Huntington's disease,dystonia, Leber's hereditary optic neuropathy, schizophrenia, andmyodegenerative disorders such as “mitochondrial encephalopathy, lacticacidosis, and stroke” (MELAS), and “myoclonic epilepsy ragged red fibersyndrome” (MERRF). For example, a vector capable of expressing theprotein of SEQ ID NO:72, or biologically active fragments thereof, canbe administered to a subject to treat or prevent disorders including,but not limited to, those described above. Alternatively, the vector canencode a variant, or biologically active fragment of the variantprotein. Multiple vectors encoding any combination of SEQ ID NO:72,variants, and/or biologically active fragments of SEQ ID NO:72 and/orvariants can be administered to a subject.

The invention relates to methods and compositions using the protein ofthe invention or fragment thereof to deacetylate substrates, alone or incombination with other substances, for example, but not limited tosilence specific target genes. Acetylated substrates used in suchmethods are preferably acetylated histones and acetyltransferases. Forexample, the protein of the invention or fragment thereof is added to asample containing a substrate in conditions allowing deacetylation, andallowed to catalyze the deacetylation of the substrate. In a preferredembodiment, the deacetylation is carried out using a standard assay suchas those described in Landry and collaborators [Landry et al., Proc.Natl. Acad. Sci. 97:5807-5811 (2000), the disclosure of which isincorporated by reference in its entirety]. Deacetylated histonesobtained by this method may be mixed with purified naked DNA (plasmidpreparations for example) in order to reconstitute chromatine-likestructures in vitro. Such structures are of great interest in the studyof enzymatic factors involved in transcription and replication. Naturaltranscription factors are unable to enter the condensed chromatin, andthe gene function is effectively switched-off. Also, the chromatincondensation constitutes a valuable parameter in the assessment of malefertility, completely independent of conventional sperm parameters[Hammadeh, M. E., et al., Arch Androl;46(2):99-104 (2001)].

Another embodiment of the present invention relates to composition andmethods of using the protein of the invention or fragment thereof toscreen for inhibitors and activators of deacetylase activity. Suchdeacetylase inhibitors are of great potential as new drugs due to theirability to influence transcriptional regulation and to induce apoptosisor differentiation in cancer cells [Marks P. A. et al., Clin. CancerRes. 7:759-760 (2001)], and also as antiproliferative reagents involvedin antiprotozoal, antifugal, phytotoxic and antiviral applications[Meinke, P. T. & Liberator, P., Curr. Med. Chem. 8:211-235 (2001)]. Inone such embodiment, the protein of the invention is contacted in vitrowith a fluorescently labeled acetylated substrate as well as a testagent, and the activity of the protein is detected, wherein a differencein the activity of the protein in the presence of the test agent incomparison to the activity in the absence of the test agent indicatesthat the test agent is a modulator of the protein. Suitable substratesinclude, e.g., aminocoumarin derivative of an acetylated lysine, whichcan be quantitated using a reverse-phase HPLC-system with a fluorescencedetector [see, e.g., Hoffmann et al., Nucl. Acids Res. 27:2057-2058(1999); Hoffmann et al., Pharmazie 55:601-606 (2000); the disclosures ofeach of which are incorporated herein in their entireties].

In another preferred embodiment, the polynucleotides of SEQ ID:71,polypeptides of SEQ ID:72 or antibodies to the polypeptide of thepresent invention may also be used in screening methods for detecting anabnormally decreased or increased level of polypeptides or mRNA, as wellas to detect the effect of added compounds on the production of thepresent mRNA and polypeptide in cells. Abnormal activity of our proteinis associated with accelerated aging syndromes such as Cochayne'ssyndrome, Ataxia telangiectasia and Werner's syndrome as well asage-associated diseases as well as “early onset” forms of diseasesassociated with old age such as dementia and Parkinson's disease.Decreased or increased expression can be measured, for example, at theRNA level using any of the methods known in the art for thequantification of polynucleotides, such as nucleic acid amplificationmethods including PCR and RT-PCR, as well as RNAse protection, Northernblotting and other hybridization methods. Expression can also bedetected using assays to determine levels of the present protein, suchas ELISA assays. These methods can also be used to discover agents whichinhibit or enhance the production of polypeptide in cells or tissues.Examples of potential polypeptide inhibitors include antibodies,oligonucleotides, heterologous proteins, or small molecule inhibitors ofthe present protein.

Another embodiment of the invention relates to methods of preparingantibodies that selectively bind to the protein of the invention orfragment thereof. Such antibodies may be used, for example, inco-immunoprecipitation procedures that enrich for chromatin fragmentscontaining binding sites for the protein of the invention. This methodmay identify genes or regions of the human genome silenced by thedeacetylase activity of the protein of the invention and also proteinswhich interact with the compacted form of the chromatin like RCC1(regulator of chromosome condensation) [Renault, L. et al., Cell,105:245-255 (2001)]. For example, in one method, antibodies thatselectively bind to HDAC are coupled to protein A or protein G sepharosebeads and added to samples containing fragments of native chromatinunder conditions amenable to immunoprecipitation, and the DNA fragmentsco-precipitated with HDAC are extracted and subcloned. These DNAfragments can then be either sequenced and/or used as probes to screengenomic libraries [Gould et al., Nature 348:308-312 (1990), thedisclosure of which is incorporated herein by reference in itsentirety].

In another embodiment, the invention relates to methods and compositionsusing the protein of the invention or fragment thereof as a markerprotein to selectively identify tissues, such as salivary gland orplacenta, or to distinguish between two or more possible sources of atissue sample on the basis of the level of the protein of SEQ ID NO:72in the sample. For example, the protein of SEQ ID NO:72 or fragmentsthereof may be used to generate antibodies using any techniques known tothose skilled in the art, and the antibodies may then be used toidentify tissues of unknown origin, for example, forensic samples,differentiated tumor tissue that has metastasized to foreign bodilysites, or to differentiate different tissue types in a tissuecross-section using immunochemistry. Typically, in such methods a tissuesample is contacted with the antibody, which may be detectably labeled,under conditions which facilitate antibody binding. In one embodiment,the level of antibody binding to the test sample is measured andcompared to the level of binding expected from control cells fromsalivary gland and placenta, or tissues other than salivary gland andplacenta, to determine whether the test sample is from salivary glandand placenta. Such methods may also be performed in conjunction withother, independant methods for determining cellular identity. Similarmethods can be used to specifically detect cells expressing the protein,as well as to specifically isolate cells expressing the protein or toisolate the protein itself. For example, an antibody against the proteinof SEQ ID NO:72 or a fragment thereof may be fixed to a solid support,such as a chromatography matrix. A preparation containing cellsexpressing the protein of SEQ ID NO:72 is placed in contact with theantibody under conditions which facilitate binding to the antibody. Thesupport is washed and then the protein is released from the support bycontacting the support with agents which cause the protein to dissociatefrom the antibody.

A preferred embodiment of the invention relates to compositions ormethods using the protein of SEQ ID NO:72 or fragment thereof todiagnose, treat and/or prevent disorders caused by the expression ofgenes whose transcription is regulated by the extent of local chromatincondensation. The number of pathologies and conditions that could betreated by the protein of the invention is potentially huge andunlimited. Favored disorders linked to dysregulation of genetranscription such as cancer and other disorders relating to abnormalcellular differentiation, proliferation, or degeneration, includingleukemia, lymphomas, prostate hypertrophy, kidney diseases, kidneyfailures, viral infection especially HIV and viral hepatitis (i.e.expression of viral proteins), metabolic diseases such as obesity and anumber of inflammatory diseases, for example due to interleukinover-expression. For diagnostic purposes, the expression of the proteinof the invention can be investigated using any method, for exampleNorthern blotting, RT-PCR or immunoblotting methods, and compared to theexpression in control individuals. For prevention and/or treatmentpurposes, the expression of the protein of the invention may beenhanced, inhibited, or otherwise altered in a patient using any of anumber of methods, including gene therapy methods, or by administering acompound that enhances or inhibits the expression or activity of theprotein.

In one embodiment, the present invention provides a method forinhibiting the proliferation of a cell, the method comprisingintroducing into the cell the protein of the invention, linked to aheterologous protein domain that specifically targets the presentprotein to a cell-proliferation-regulating gene, wherein the targetingof the present protein to the gene results in local chromatincondensation and an inhibition in the expression of the gene.Cell-fusion proteins containing both the deacetylase activity and thespecific DNA binding domain are obtained by methods of molecular biologywell known to those skilled in the art. In one embodiment, such fusionproteins are introduced into the cell by transfecting the cell with apolynucleotide encoding the fusion protein, wherein the fusion proteinis expressed in the cell. Such polynucleotides, e.g. in the form ofexpression vectors, which can thus be used, e.g., for gene therapy totreat or prevent cancer, metabolic disorders, aging and any disorderwhere a gene is over-expressed in association with local chromatindecondensation. Such recombinant cDNA may be introduced, for example,using in any vector, viral or non-viral, and viral vectors can be butnot limited to retroviral, adenoviral, and adeno-associated vectors,which have been used in cancer therapy (Alemany et al., Nat. Biotechnol.18:723-727 (2000)). Another approach is to administer a therapeuticamount of a polypeptide of SEQ ID:72, preferably in combination with asuitable pharmaceutical carrier. Such carriers include, but are notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanoland combinations thereof.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of SEQ ID NO:71 or fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations.The microarray can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents (see for example: Chee, M. et al., Science,274:610-614 (1996)). It has been shown that multiple classical featuresof cancer cells can be manifested by improper histone deacetylation [forreview see Wade, P. A. Hum Mol Genet;10(7):693-698 (2001)].

Another related embodiment relates to the use of SEQ ID NO:72, itscomplement, or any part thereof to develop antagonists of the protein ofthe invention and of the HDAC complex. Antagonists or inhibitors ofhistone deacetylase may indeed be used to suppress gene silencing. Suchantagonists and/or inhibitors may be antibodies specific for the proteinof the invention that can be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express the protein of theinvention. Other methods to inhibit the expression of the protein of theinvention include antisense and triple helix stategies as describedherein. Other antagonists or inhibitors of the protein of the inventionmay be produced using methods which are generally known in the art,including the screening of libraries of pharmaceutical agents toidentify those which specifically bind the protein of the invention. Theprotein of the invention, or fragment thereof, preferably its functionalor immunogenic fragments, or oligopeptides related thereto, can be usedfor screening libraries of compounds in any of a variety of drugscreening techniques. The fragment employed in such screening may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenthe protein of the invention, or fragment thereof, or derivativethereof, and the agent being tested, may be measured. Another techniquefor drug screening which may be used provides for high throughputscreening of compounds having suitable binding affinity to the proteinof the invention as described in published PCT application WO84/03564.Abnormal gene silencing causes conditions like, but not limited to,accelerated aging syndromes such as Cochayne's syndrome, Ataxiatelangiectasia and Werner's syndrome as well as age-associated diseasesas well as “early onset” forms of diseases associated with old age suchas dementia and Parkinson's disease.

Protein of SEQ ID:74 (Internal Designation Clone500716683_(—)204-24-2-0-D12-F)

The protein of SEQ ID NO:74, herein referred as short Paraplegin,comprising the amino acid sequence:

MAVLLLLLRALRRGPGPGPRPLWGPGPAWSPGFPARPGRGRPYMASRPPGDLAEAGGRALQSLQLRLLTPTFEGINGLLLKQHLVQNPVRLWQLLGGTFYFNTSRLKQKNKEKDKSKGK APEEDEGIFI,is encoded by the cDNA of clone 500716683_(—)204-24-2-0-D12-F (SEQ IDNO:73). Accordingly, it will be appreciated that all characteristics anduses of the polypeptide of SEQ ID NO:74 described throughout the presentapplication also pertain to the polypeptide encoded by a nucleic acidincluded in clone 500716683_(—)204-24-2-0-D12-F. In addition, it will beappreciated that all characteristics and uses of the nucleic acid of SEQID NO:73 described throughout the present application also pertain tothe nucleic acid included in clone 500716683_(—)204-24-2-0-D12-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:73, SEQ ID NO:74, and Clone500716683_(—)204-24-2-0-D12-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:74 is encoded by a nucleic acid of 879nucleotides with an ORF between nt 9 to 395 yielding a 129 amino acidprotein. The protein is a variant of the sequence for human protease andassociated protein-15 (PPRG-15) (described in PCT publicationWO200009709-A2, the disclosure of which is incorporated herein byreference in its entirety) and of the sequence of the protein associatedto hereditary spastic paraplegia (described in PCT publicationWO9958556-A2, the disclosure of which is incorporated herein byreference in its entirety). It has a signal peptide spanning 17 aminoacid residues at its N-terminal. The protein of SEQ ID NO:74 islocalized in the brain and has a mitochondrial localizing signalpeptide.

Moreover, the protein of SEQ ID NO:74 exhibits high homology to theN-terminal of hereditary spastic paraplegia protein sequence (describedin PCT publication WO9958556-A2). Hereditary Spastic paraplegia (HSP) ischaracterized by progressive weakness and spasticity of the lower limbsdue to degeneration of corticospinal axons. (Harding, A. E., J. Med.Genet. 18: 436-441(1981); Fink, J. K., et al., Am. J. Hum. Genet.56:188-192 (1997); Reid, E., J. Med. Genet. 34:499-503, (1997)). This isa genetically heterogeneous group of neurodegenerative disordersaffecting approximately 1 in 10,000 individuals (Filla (1992); Polo etal. (1993)). Patients with HSP typically show leg stiffness and gaitdisturbance, decreased perception of sharp stimulation, and diminishedvibratory sense in the distal lower limbs. Both the age of onset andseverity of the symptoms are highly variable even among individuals fromthe same family (Harding, A. E., J. Med. Genet. 18: 436-441(1981); Dürret al., 1994). Currently, no specific treatment is available to prevent,cure, or delay progression of symptoms of HSP.

In addition to the above-described clinical spectrum, which is typicalof the “pure” form of HSP, several patients have been shown to have“complicated” forma of HSP characterized by the presence of additionalneurological and non-neurological symptoms such as metal retardation,peripheral neuropathy, amyotrophy, ataxia, retinitis pigmentosa, opticatrophy, deafness, and ichtyosis (Bonneau, D., et al., J. Med. Genet.30:381-384 (1993); Gigli, G. L., et al., Am. J. Med. Genet. 45:711-716(1993); Lizcano-Gil, L. A. et al., Am. J; Med. Genet. 68:1-6 (1997);Webb, S., et al., Epilepsia 38:495-499 (1997)). Albeit some of theseforms have been found to segregate in families, it is still unclearwhether complicated forms of HSP represent distinct genetic entities orvariant presentations of pure HSP. However, even in pure forma of HSP(i.e., with clinical features limited to the lower segments), a broadersubclinical involvement of the nervous system has been demonstrated[Tedeschi, G. et al., J. Neurol. Sci. 103:55-60 (1991); Dürr, A., etal., Neurology 44:1274-1277 (1994)].

Autosomal dominant, autosomal recessive, and X-linked forms of HSP havebeen described, indicating genetic heterogeneity [Harding, A. E., J.Med. Genet. 18: 436-441(1981); Fink, J. K., et al., Am. J. Hum. Genet.56:188-192 (1997); Reid, E., J. Med. Genet. 34:499-503, (1997)]. Casariand collaborators have identified and characterized a gene associated tohereditary spastic paraplegia, located in the telomere region ofchromosome 16q, and the protein deriving, therefrom, named paraplegin[Casari, G., et al. Cell 93:973,983 (1998)].

It is believed that the protein of SEQ ID NO:74, or fragment thereof isa mitochondrial protein associated to hereditary spastic paraplegia. Theprotein of the invention or fragment thereof may play a role in themitochondrial degradation machinery. Preferred polypeptides of theinvention are polypeptides comprising the amino acids of SEQ ID NO:74from positions 1 to 125. Other preferred polypeptides of the inventionare fragments of SEQ ID NO:74 having any of the biological activitiesdescribed herein.

Another embodiment of the invention relates to compositions and methodsusing the protein of the invention or fragment thereof to labelmitochondria in order to visualize any change in number, topology ormorphology of this organelle, for example in association with amitochondria-related human disorder, such as hereditary spasticparaplegia [Casari, G., et al. Cell 93:973,983 (1998)], neurolepticmalignant syndrome (NMS) [Kubo et al., Forensic Sci. Int. 115:155-158(2001)], the Rett syndrome [Armstrong, Brain Dev. 14 Suppl:S89-98(1992)], Alpers disease [Chow and Thorburn, Hum. Reprod. 15 Suppl2:68-78 (2000)] or mitochondrial encephalomyopathies [Handran et al.,Neurobiol. Dis. 3:287-298 (1997)]. Casari and collaborators have shownthat paraplegin protein localizes to mitochondria by immunofluorescencestudies [Casari, G., et al. Cell 93:973,983 (1998)]. Paraplegin proteinexhibits a helical wheel pattern (an amphiphilic structure composed ofbasic residues, mainly arginine, on one side and apolar residues on theopposite side) of the N-terminal which is highly homolog to the proteinof the invention; moreover the high ratio of arginine to lysin among thefirst 41 amino acids indicates the presence of typical mitochondrialleader sequences [Casari, G., et al. Cell 93:973,983 (1998)]. Forexample, the protein may be rendered easily detectable by inserting thecDNA encoding the protein of the invention into a eukaryotic expressionvector in frame with a sequence encoding a tag sequence. Eukaryoticcells expressing the tagged protein of the invention may also be usedfor the in vitro screening of drugs or genes capable of treating anymitochondria-related disease or conditions. Another example, the proteinof the invention or fragment thereof may be used to generate specificantibodies which would in turn allow the visualization of mitochondrialstructures by methods well-known to those of skill in the art.

In another embodiment, the protein of the invention may be used totarget heterologous compounds (polypeptides or polynucleotides) to thebrain and/or the mitochondria. For instance, a chimeric protein composedof the protein of the invention recombinantly or chemically fused to aprotein or polynucleotide of therapeutic interest would allow thedelivery of the therapeutic protein/polynucleotide specifically to theabove-mentioned cellular/tissue targets (mitochondria, brain). Preferredfragments are the putative peptide signal, and/or any other fragments ofthe protein of the invention that may contain targeting signals formitochondria). Such heterologous compounds may be used to modulatemitochondrial activities, such as to induce and/or preventmitochondrial-induced apoptosis or necrosis. For example, theseheterologous compounds may be used in the treatment and/or theprevention of disorders due to mitochondrial dysfunction, including, butnot limited to, hereditary spastic paraplegia. In addition, heterologouspolynucleotides may be used to deliver nucleic acids for mitochondrialgene therapy, i.e. to replace a defective mitochondrial gene and/or toinhibit the deleterious expression of a mitochondrial gene.

An antagonist of the protein of SEQ ID NO:74 may be produced usingmethods which are generally know in the art. In one aspect, the proteinof the invention or fragment may be used to synthesize specificantibodies using any techniques known to those skilled in the artincluding those described therein. In particular, purified shortparaplegin may be used to produce antibodies or to screen libraries ofpharmaceutical agents to identify those which specifically bind shortparaplegin.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified protein of SEQ ID NO:74 in conjunction with asuitable pharmaceutical carrier may be administered to a subject totreat or prevent a disorder associated with change of expression oractivity of short paraplegin including, but not limited to, thosedescribed above. The antibody which specifically binds short parapleginmay be used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissues which express short paraplegin.

In another embodiment, antibodies which specifically bind the protein ofSEQ ID NO:74 may be used for the diagnosis of disorders characterized byexpression of short paraplegin. Truncated forms of paraplegin areinvolved in hereditary spastic paraplegia (Casari, G., et al. Cell93:973,983 (1998)). Diagnostics assays for short paraplegin includemethods which utilize the antibody and a label to detect shortparaplegin in human body fluids or in extract of cells or tissues. Avariety of protocols for measuring short paraplegin, including ELISA's,RIAs, and FACs, are known in the art and provide a basis for diagnosingthe presence of short paraplegin expression.

In another embodiment, the polynucleotide of SEQ ID NO:73 or a fragmentmay be used for diagnostic purposes in assays that detect the presenceof associated disorders, for example but not limited to,. hereditaryspastic paraplegia. The polynucleotides which may be used includeoligonucleotide sequences, complementary RNA and DNA molecules, PNAs.The polynucleotides may be used to detect and quantitate gene expressionin biopsied tissues in which expression of short paraplegin may becorrelate with disease. The nucleotide sequences encoding shortparaplegin may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly increased in comparison to a control sample then thepresence of increased levels of nucleotide sequences encoding shortparaplegin in the sample indicates the presence of associated disorder,particularly but not limited to, hereditary spastic paraplegia. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orto monitor the treatment of an individual patient.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period of time.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of SEQ ID NO:73 or fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations.The microarray can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents [see for example: Chee, M. et al., Science,274:610-614 (1996)]. For example, it has been shown that geneticvariants, mutations, and polymorphisms are related to hereditary spasticparaplegia [for review see Casari, G., and Rugarli, E. Curr. Opin.Genetics and Development, 11:336-342 (2001)].

In another preferred embodiment, the protein of the invention orfragment thereof can be used in an enzyme/prodrug strategy to treat anumber of pathologies, especially those treated with drugs associatedwith severe side effects, including, but not limited to, autoimmunediseases and chronic inflammatory diseases such as rheumatoid arthritis,and cancer chemotherapy. These side effects can be mainly explained bythe fact that the in vivo selectivity of the drugs used is too low (forexample, the inadequate selectivity between tumor and normal cells ofmost anticancer drugs is well known and their toxicity to normal tissuesis dose limiting). In the first phase of one example of such a protocol,a conjugate of the protein of the invention or fragment thereof and anantibody to a tissue specific antigen (for example, tumor specificantigens in the case of cancer chemotherapy) is administered. After adelay to allow residual enzyme conjugate to be cleared from the blood, arelatively non-toxic compound is administered to the patient. Thisnon-toxic compound is a substrate of the protein of the invention, andis converted by the protein into a substantially more toxic compound.Thus, because of the previous, targeted administration of the protein ofthe invention, when the non-toxic compound is administered, the toxiccompound is only produced in the vicinity of the cells targeted by thefusion protein. This two-phase approach has been termedantibody-directed enzyme-prodrug therapy (ADEPT), this approach isreviewed by Melton et al. [Melton R. et al., J. Natl. Cancer Inst., 88,p 153-165 (1996)]. Alternatively the first phase can be replaced by agene therapy approach resulting in the de novo synthesis of the proteinof the invention or fragment thereof by cells from the targeted tissue,this has been termed gene-dependent enzyme/prodrug therapy (GDEPT).Another advantage of these 2 approaches (ADEPT and GDEPT) is that asingle enzyme molecule is capable of activating many prodrug molecules.

Protein of SEQ ID:76 (Internal Designation Clone500760207_(—)205-58-4-0-H6-F)

The protein of SEQ ID NO:76, herein referred as Ketothiolase (KT),comprising the amino acid sequence:

MMGVFVVAAKRTPFGAYGGLLKDFTATDLSEFAAKAALSAGKVSPETVDSVIMGNVLQSSSDAIYLARHVGLRVGIPKETPALTINRLCGSGFQSIVNGCQEICVKEAEVVLCGGTESMSQAPYCVRNVRFGTKLGSDIKLEDSLWVSLTDQHVQLPMAMTAENLAVKHKISREECDKYALQSQQRWKAANDAGYFNDEMAPIEVKTKKGKQTMQVDEHARPQTTLEQLQKLPPVFKKDGTVTAGNASGVADGAGAVIIASEDAVKKHNFTPLARIVGYFVSGCDPSIMGIGPVPAISGALKKAGLSLKDMDLVEVNEAFAPQYLAVERSLDLDISKTNVNGGAIALGHPLGGSGSRITAHLVHELRRRGGKYAVGSACIGGGQGIAVIIQSTA, is encoded by the cDNA of clone500760207_(—)205-58-4-0-H6-F (SEQ ID NO:75). Accordingly, it will beappreciated that all characteristics and uses of the polypeptide of SEQID NO:76 described throughout the present application also pertain tothe polypeptide encoded by the human cDNA of clone500760207_(—)205-58-4-0-H6-F. In addition, it will be appreciated thatall characteristics and uses of the nucleic acid of SEQ ID NO:75described throughout the present application also pertain to the humancDNA of clone 500760207_(—)205-58-4-0-H6-F. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:75, SEQID NO:76, and Clone 500760207_(—)205-58-4-0-H6-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

The protein of SEQ ID NO:76 encoded by the cDNA of SEQ ID NO:75 is apolymorphism variant of 3-Ketoacyl CoA Thiolase protein (GENPEPTaccession number D16294). Furthermore, a BLAST search with the aminoacid sequence of SEQ ID NO:76 indicates that the protein of theinvention is homologous to 3-Ketoacyl CoA thiolase of rat (Swissprotaccession number P13437) and Bacillus halodurans (Genbank accessionnumber AP001514).

The 394 amino acids protein of SEQ ID NO:76 displays 1 candidatemembrane-spanning segment, from amino acids 373 to 393. Accordingly,some embodiments of the present invention relate to polypeptidescomprising the transmembrane domain. Finally, the protein of theinvention displays the 3 thiolase signatures (PS00098, PS00737, PS00099)spanning from positions 85 to 103, positions 339 to 355, and positions374 to 387, respectively. Accordingly, some embodiments of the presentinvention relate to polypeptides comprising the thiolase signature.

Living organisms are exposed to a number of different fatty acids andtheir various derivatives arising either via endogenous synthesis orfrom exogenous sources. These hydrophobic compounds can play specificmetabolic, structural or endocrinic functions in the organisms beforetheir elimination, which can be metabolism to CO₂ or to more polar lipidmetabolites allowing their excretion. Quantitatively, one of the majorpathways metabolizing fatty acids is β-oxidation, which is oftendescribed as a spiral of four reactions catalyzed by three enzymes.

The three consecutive steps of mitochondrial β-oxidation of fatty acids,including the long-chain 3-hydroxyl-CoA dehydrogenase, are catalyzed bythe trifunctional protein: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoAdeshydrogenase and 3-ketoacyl-CoA thiolase. Deficiencies in enzymeactivities of the heterocomplex, which contains 4 alpha and 4 betasubunits, causes sudden unexplained infant death, a Reye-like syndrome,cardiomyopathy, or skeletal myopathy.

Defects in the trifunctional protein fall into two groups: patients withan isolated defect in 3-hydroxyacyl-CoA dehydrogenase and those with adeficiency in all three activities and absence of immunoreactive protein(Tyni, J. et al., Acta Paeditr. 88:237-245 (1999)). Patients in thesecond group have been found to have either deletions in the α-subunitcDNA encoding for 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoAdeshydrogenase or point mutations in the β-subunit encoding for3-ketoacyl-CoA thiolase [Ushikubo, S. et al., Am. J; Hum. Genet. 58:979-988 (1996); Ori, K. F. et al., Hum. Mol Genet. 6: 1215-1224 (1997)].

It is believed that the protein of SEQ ID NO:76 or fragment thereof isan hydrolase, preferably acting on ester bonds, more preferably athiolester hydrolase, even more preferably an ketoacyl-CoA thiolasewhich, as such, plays a role in fatty acid metabolism, in cellularvesicle transport and maintenance of the cytoarchitecture, in cellularproteolysis, endocytosis, signal transduction, lysosomal storage, cellproliferation and differentiation, immune and inflammatory response. Theenzyme's substrates are compounds preferably containing an ester bond,preferably a thiol ester bond, more preferably an acyl thioester bond.Preferred polypeptides of the invention are polypeptides comprising theamino acids of SEQ ID NO:76 from positions 85 to 103, positions 339 to355, and positions 374 to 387. Other preferred polypeptides of theinvention are fragments of SEQ ID NO:76 having any of the biologicalactivities described herein. The hydrolytic activity of the protein ofthe invention or fragment thereof may be assayed using any of the assaysknown to those skilled in the art including those described in U.S. Pat.No. 5,445,942. The ability to bind a cofactor may also be assayed usingany techniques well known to those skilled in the art including, forexample, the assay for binding NAD described in U.S. Pat. No. 5,986,172.

Another embodiment of the invention relates to compositions and methodsusing the protein of the invention or fragment thereof to labelmitochondria, or more specifically the inner mitochondrial membrane, inorder to visualize any change in number, topology or morphology of thisorganelle, for example in association with a mitochondria-related humandisorder, such as neuroleptic malignant syndrome (NMS) (Kubo et al.,Forensic Sci. Int. 115:155-158 (2001)), the Rett syndrome (Armstrong,Brain Dev. 14 Suppl:S89-98 (1992)), Alpers disease (Chow and Thorburn,Hum. Reprod. 15 Suppl 2:68-78 (2000)) or mitochondrialencephalomyopathies (Handran et al., Neurobiol. Dis. 3:287-298 (1997)).For example, the protein may be rendered easily detectable by insertingthe cDNA encoding the protein of the invention into a eukaryoticexpression vector in frame with a sequence encoding a tag sequence.Eukaryotic cells expressing the tagged protein of the invention may alsobe used for the in vitro screening of drugs or genes capable of treatingany mitochondria-related disease or conditions.

In one embodiment, the invention relates to compositions and methodsusing the protein of SEQ ID NO:76 or fragment thereof as a marker fortissue types (especially placenta), or to distinguish between two ormore possible sources of a tissue sample on the basis of the level ofthe protein of SEQ ID NO:76 in the sample. For example, the protein ofSEQ ID NO:76 or fragments thereof may be used to generate antibodiesusing any techniques known to those skilled in the art, and theantibodies may then be used to identify tissues of unknown origin, forexample, forensic samples, differentiated tumor tissue that hasmetastasized to foreign bodily sites, or to differentiate differenttissue types in a tissue cross-section using immunochemistry. Typically,in such methods a tissue sample is contacted with the antibody, whichmay be detectably labeled, under conditions which facilitate antibodybinding. In one embodiment, the level of antibody binding to the testsample is measured and compared to the level of binding expected fromcontrol cells from placenta, or tissues other than placenta to determinewhether the test sample is from placenta. Such methods may also beperformed in conjunction with other, independant methods for determiningcellular identity. Similar methods can be used to specifically detectcells expressing the protein, as well as to specifically isolate cellsexpressing the protein or to isolate the protein itself. For example, anantibody against the protein of SEQ ID NO:76 or a fragment thereof maybe fixed to a solid support, such as a chromatography matrix. Apreparation containing cells expressing the protein of SEQ ID NO:76 isplaced in contact with the antibody under conditions which facilitatebinding to the antibody. The support is washed and then the protein isreleased from the support by contacting the support with agents whichcause the protein to dissociate from the antibody.

In another embodiment, the protein of the invention may be used totarget heterologous compounds (polypeptides or polynucleotides) to theplacenta and/or the cell mitochondria. For instance, a chimeric proteincomposed of the protein of the invention recombinantly or chemicallyfused to a protein or polynucleotide of therapeutic interest would allowthe delivery of the therapeutic protein/polynucleotide specifically tothe above-mentioned cellular/tissue targets (mitochondria, placenta).

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention orfragment thereof to establish transgenic model animals (D. melanogaster,M. musculus), by any method familiar to those skilled in the art. Bymodulating in vivo the expression of the transgene with drugs ormodifier genes (activator or suppressor genes), animal models can bedeveloped that mimic human mitochondria-associated disorders such asmyopathies or obesity. These animal models would thus allow theidentification of potential therapeutic agents for treatment of thedisorders. In addition, recombinant cell lines derived from thesetransgenic animals may be used for similar approaches ex vivo.

In another embodiment, the invention relates to compositions and methodsusing the proteins of the invention or fragment thereof such as ligandsfor substrates of interest. In a preferred embodiment, the proteins ofthe invention or fragment thereof may be used to identify and/orquantify substrates using any techniques known to those skilled in theart. To find substrates, the proteins of the invention, or fragmentthereof, or derivative thereof, may be used for screening libraries ofcompounds in any of a variety of drug screening techniques. The fragmentemployed in such screening may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Theformation of binding complexes, between the proteins of the invention,or fragment thereof, or derivative thereof, and the agent being tested,may be measured. Antagonists or inhibitors of the proteins of theinvention may be produced using methods which are generally known in theart, including the screening of libraries of pharmaceutical agents toidentify those which specifically bind the protein of the invention.Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe proteins of the invention as described in published PCT applicationWO84/03564.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with an altered levelsof the protein of the invention like, but not limited to, deficiency ofthe hydrogenase activity (LCHAD deficiency). Diagnostic assays to detectthe protein of the invention may comprise a biopsy, in situ assay ofcells from organ or tissue sections, or an aspirate of cells from atumor or normal tissue. In addition, assays may be conducted uponcellular extracts from organs, tissues, cells, urine, or serum or bloodor any other body fluid or extract.

Assays for the quantification of the KT of SEQ ID NO:76 may be performedaccording to methods well known in the art. Typically, these assayscomprise contacting the sample with a ligand of the protein of theinvention or an antibody (polyclonal or monoclonal) which recognizes theprotein of the invention or a fragment thereof, and detecting thecomplex formed between the protein of the invention present in thesample and the ligand or antibody. Fragments of the ligands andantibodies may also be used in the binding assays, provided thesefragments are capable of specifically interacting with the KT of thesubject invention. Further, the ligands and antibodies which bind to theKT of the invention may be labeled according to methods known in theart. Labels which are useful in the subject invention include, but arenot limited to, enzymes labels, radioisotopic labels, paramagneticlabels, and chemiluminescent labels. Typical techniques are described byKennedy, J. H., et al. (1976) Clin. Chim. Acta 70:1-31; and Schurs, A.H. et al. (1977) Clin. Chim. Acta 81: 1-40.

In another embodiment, the present invention includes the use of theprotein of SEQ ID NO:76, or fragments having a desired biologicalactivity to treat or ameliorate a condition in an individual. Forexample, the condition may be deficiency of the hydrogenase activity(LCHAD deficiency), hypoglycemia, musculr hypotonia, hyperamonia, mildliver dysfunction, 3-hydrixydicarboxylic aciduria, cardiomyopathy,retinal dystrophy, Bannayan-Riley-Ruvalcaba syndrome, or an abnormalityin any of the functions of the hydrogenase activity. In suchembodiments, the protein of SEQ ID NO:76, or a fragment thereof, isadministered to an individual in whom it is desired to increase ordecrease any of the activities of the protein of SEQ ID NO:76. Theprotein of SEQ ID NO:76 or fragment thereof may be administered directlyto the individual or, alternatively, a nucleic acid encoding the proteinof SEQ ID NO:76 or a fragment thereof may be administered to theindividual. Alternatively, an agent which increases the activity of theprotein of SEQ ID NO:76 may be administered to the individual. Suchagents may be identified by contacting the protein of SEQ ID NO:76 or acell or preparation containing the protein of SEQ ID NO:76 with a testagent and assaying whether the test agent increases the activity of theprotein. For example, the test agent may be a chemical compound or apolypeptide or peptide. Alternatively, the activity of the protein ofSEQ ID NO:76 may be decreased by administering an agent which interfereswith such activity to an individual. Agents which interfere with theactivity of the protein of SEQ ID NO:76 may be identified by contactingthe protein of SEQ ID NO:76 or a cell or preparation containing theprotein of SEQ ID NO:76 with a test agent and assaying whether the testagent decreases the activity of the protein. For example, the agent maybe a chemical compound, a polypeptide or peptide, an antibody, or anucleic acid such as an antisense nucleic acid or a triple helix-formingnucleic acid.

In another embodiment, the invention also relates to the use ofpolynucleotides of SEQ ID NO:75 as diagnostic reagents. Detection of amutated form of the gene characterized by the polynucleotide of SEQ IDNO:75 which is associated with a dysfunction will provide a diagnostictool that can add to, or define, a diagnosis of a disease, orsusceptibility to a disease. It has been shown previously that mutationsin the beta subunit are responsible for trifunctional protein relateddiseases like those listed above [Ushikibo, S., et al., Am. J. Hum.Genet. 58:979-988 (1996); Ori, K. F. et al., Hum. Mol Genet. 6:1215-1224(1997)]. Individuals carrying mutations in the gene may be detected atthe DNA level by a variety of techniques known by those skilled in theart. Nucleic acids for diagnosis may be obtained from a subject cells,such as from blood, urine, saliva, tissue biopsy or autopsy material.The genomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of SEQ ID NO:75 or fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations.The microarray can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents (see for example: Chee, M. et al., Science,274:610-614 (1996)).

Protein of SEQ ID NO:78 (Internal Designation Clone122421_(—)105-076-4-0-H1-F)

The cDNA of clone 122421_(—)105-076-4-0-H1-F (SEQ ID NO:77) encodes theprotein of SEQ ID NO:78, comprising the amino acid sequence:

MAAALFVLLGFALLGTHGASGAAGTVFTTVEDLGSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPMGTANIQLHGPPRVKAVKSSEHINEGETAMLVCKSESVPPVTDWAWYKITDSEDKALMNGSESRFFVSSSQGLSELHIENLNMEADPGQYRCNGTSSKGSDQAIITLRVRSHLAALWPFLGIVAEVLVLVTIIFIYEKRRKPEDVLDDDDAGSAPLKSSGQHQNDKGKNVRQRNSS. Accordingly, it will be appreciated thatall characteristics and uses of the polypeptide of SEQ ID NO:78described throughout the present application also pertain to thepolypeptide encoded by the nucleic acids included in clone122421_(—)105-0764-0-H1-F. In addition, it will be appreciated that allcharacteristics and uses of the nucleic acid of SEQ ID NO:77 describedthroughout the present application also pertain to the nucleic acidsincluded in clone 122421_(—)105-076-4-0-H1-F. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:77, SEQID NO:78, and Clone 122421_(—)105-0764-0-H1-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

The protein of SEQ ID NO:78 (BASI2) is a novel polymorphic variant ofhuman basigin. BASI2 displays a signal peptide (MAAALFVLLGFALLGTHG), andtwo immunoglobulin (Ig) domains

(GSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVF andGETAMLVCKSESVPPVTDWAWYKITDSEDKALMNGSESRFFVSSSQGLSELHIENLNMEADGQYRCNGTSS). Furthermore, BASI2 displays three N-glycosylation sites(NDSA, NGSE, and NGTS). The arginine at position 166 in basigin ischanged to leucine in BASI2. Thus, the polymorphic, nonconservativechange present in BASI2 is located in the second Ig domain, which isinvolved in protein-protein interactions. Such a polymorphic changelocated in the second Ig domain has never been previously reported.Thus, as a novel polymorphic variant of basigin, BASI2 displays similarbiological activities as basigin, but displays enhanced kineticparameters during protein-protein interactions.

BASI2 is a member of the immunoglobulin superfamily, which includes Tcell receptors, neural cell adhesion molecules and majorhistocompatibility complex antigens. BASI2 is a cell surfacetransmembrane glycoprotein that is broadly distributed, and expressed atparticularly high levels on activated gliomas, on tumor cells, onactivated T cells and at the retinal pigment epithelium and neonatalblood-brain barrier. BASI2 is involved in cell-cell interactions, andhas a multiplicity of biological roles. Notably, BASI2 stimulates thebiosynthesis of various matrix metalloproteinases (MMPs), a group ofenzymes involved in the degradation of most of the components of theextracellular matrix. In particular, MMP biosynthesis is crucial intumor secretion and in immune response. BASI2 plays a role inspermatogenesis and fertilization, in neuronal interactions in thecentral nervous system and in HIV-1 infection.

An embodiment of the present invention relates to methods of using BASI2or fragment thereof to stimulate the biosynthesis of metalloproteinases.In a preferred embodiment, metalloproteinases produced by such methodscan be used in a “cocktail” of proteases that is able to digest a widerange of proteins without knowing any of the proteins. Such proteasecocktails are useful in laboratory assays to degrade undesirableproteins in a sample, for example for removing proteins in a DNApreparation or for removing enzymes after any enzymatic reaction. Inanother preferred embodiment, metalloproteinases produced by suchmethods can be used for screening and/or assaying metalloproteinasesinhibitors. Such metalloproteinase inhibitors are very useful to treatand/or prevent a wide range of diseases associated to metalloproteinaseactivation. In still another preferred embodiment, metalloproteinasesproduced by such methods can be used for degradation of connectivetissues, for example in food industry. Any method of stimulatingmetalloproteinases biosynthesis can be used in such methods. Forexample, fibroblasts can be stimulated as described by Guo et al (J BiolChem 272:24-7 (1997)), which disclosure is hereby incorporated byreference in its entirety.

An embodiment of the present invention relates to methods of using BASI2or fragment thereof for the diagnosis of cancers, graft rejections, andgraft versus host diseases. In such methods, BASI2 or fragment thereofis used as a marker to detect and/or quantify cells in which BASI2and/or basigin expression is up-regulated, and in which MMPs aresynthetised. Any method of detecting the presence, level or activity ofBASI2 and/or basigin can be used in such methods. For example, theprotein of the invention or fragment thereof may be used to generatespecific antibodies using standard methods. Preferably, the antibodiesare either directly or indirectly labeled, and recognize the second Igdomain. In a preferred embodiment, the antibodies bind more specificallyto BASI2 than to related proteins such as basigin. Such antibodies canbe used for specifically detecting the presence of the BASI2 variant. Inanother preferred embodiment, the antibodies recognize both basigin andBASI2. Such antibodies can be used for detecting total amount of basiginand BASI2 molecules. Alternatively, the nucleic acid of the invention orfragment thereof may be used to synthesize specific probes using anytechnique known to those skilled in the art. In such assays anddiagnostic kits, the detection of a higher level of BASI2 and/or basiginexpression, compared to a control representative of a non-malignant cellcoming from a given tissue or bodily fluid, diagnostics the presence ofa tumor or the beginning of a graft rejection reaction.

Another embodiment of the present invention relates to compositions andmethods for inhibiting the activity or expression of BASI2 in a patientfor the treatment or prevention of disorders caused or aggravated as aresult of metalloproteinase biosynthesis. The inhibition of BASI2activity or expression can be achieved using any suitable method, e.g.through administration of a therapeutically effective amount of anantibody that recognizes BASI2 or fragment thereof to a patient.Preferably, the antibodies are either directly or indirectly labeled,and recognize the second Ig domain. In a preferred embodiment, theantibodies bind more specifically to BASI2 than to related proteins suchas basigin. Such antibodies can be used for specifically inhibiting theBASI2 variant. In another preferred embodiment, the antibody recognizesboth BASI2 and basigin. Such antibodies can be used for inhibiting bothisoforms. The antibody can be administrated alone or in combination withone or more agent known in the art, e.g. ABX-CBL antibody described inPCT Patent WO99/45031. Administration of the antibody can be donefollowing any method known in the art, including that described in PCTPatent WO99/45031, which disclosure is hereby incorporated by referencein its entirety. Other inhibitors of BASI2 expression or activity whichcan be used include, but are not limited to, antisense molecules,ribozymes, dominant negative forms of BASI2, and compounds that decreasethe activity or expression of BASI2 in a cell. Such compounds can bereadily identified, e.g. by screening test agents tumor cellsoverexpressing BASI2, and detecting the ability of the test agents todecrease metalloproteinase biosynthesis or to diminish the level ofBASI2 expression. Diseases and disorders caused or aggravated as aresult of MMP biosynthesis and that can be treated by administrating aninhibitor of BASI2 and/or basigin include but are limited to cancers,graft rejections and graft versus host diseases.

Still another embodiment relates to compositions and methods forinhibiting the expression or activity of BASI2 in a patient for thetreatment or prevention of disorders caused or aggravated as a result ofmicroglial activation. The inhibition of BASI2 activity or expressioncan be achieved using any of the methods described above. Disorderscaused or aggravated as a result of microglial activation include butare not limited to spinal cord contusion, Huntington disease, dementiawith Lewy bodies, ischemia, multiple sclerosis, and Alzheimer's disease.

Still another embodiment relates to compositions and methods forinhibiting the interaction between BASI2 and cyclophilin A (CyPA) in apatient, in order to treat or to reduce in severity HIV-infection.Inhibition of the interaction can be achieved using any suitable method,e.g. through administration of a therapeutically effective amount of anantibody that recognizes BASI2 or fragment thereof to a patient.Preferably, the antibodies are either directly or indirectly labeled,and recognize the first Ig domain and/or the second Ig domain.Administration of the antibodies can be performed as described above.Another embodiment of the present invention relates to compositions andmethods for enhancing the expression or activity of BASI2 and/or basiginin a patient for the treatment or prevention of disorders caused oraggravated as a result of BASI2 and/or basigin deficiency such assterility, learning and memory impairments, and retinal angiogenesis.Any method or composition enhancing the expression or activity of BASI2and/or basigin, containing BASI2 or fragment thereof, a polynucleotideencoding the protein, or a compound that increases the expression oractivity of BASI2, can be used. Such compounds can be readilyidentified, e.g. by screening test agents against non activated glialcells expressing BASI2 and detecting the ability of the test agents toenhance metalloproteinases biosynthesis, or to increase the level ofBASI2 expression. The compositions of the invention can be administereddirectly to the patient using any suitable method, for example byintravenous perfusion or by oral administration. Effective doses of thepolypeptides of the present invention are determined according to therelevant techniques.

Protein of SEQ ID NO:80 (Internal Designation 99483_(—)105-016-1-0-D7-F)

The cDNA clone 99483_(—)105-016-1-0-D7-F (SEQ ID NO:79) encodes KSPI1,the protein of SEQ ID NO:80, comprising the amino acid sequence:

MLPPPRPAAALALPVLLLLLVVLTPPPTGARPSPGPDYLRRGWMRLLAEGEGCAPCRPEECAAPRGCLAGRVRDACGCCWECANLEGQLCDLDPSAHFYGHCGEQLECRLDTGGDLSRGEVPEPLCACRSQSPLCGSDGHTYSQICRLQEAARARPDANLTVAHPGPCESGPQIVSHPYDTWNVTGQDVIFGCEVFAYPMASIEWRKDGLDIQLPGDDPHISVQFRGGPQRFEVTGWLQIQAVRPSDEGTYRCLGPMPWVKWRPLLA. Accordingly, it will be appreciated thatall characteristics and uses of the polypeptide of SEQ ID NO:80described throughout the present application also pertain to thepolypeptide encoded by the nucleic acids included in clone99483_(—)105-016-1-0-D7-F. In addition, it will be appreciated that allcharacteristics and uses of the nucleic acid of SEQ ID NO:79 describedthroughout the present application also pertain to the nucleic acidsincluded in clone 99483_(—)105-016-1-0-D7-F. A preferred embodiment ofthe invention is directed toward the compositions of SEQ ID NO:79, SEQID NO:80, and Clone 99483_(—)105-016-1-0-D7-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments Preferred KSPI1polypeptides for uses in the methods described below include thepolypeptides comprising the amino sequence of:CAPCRPEECAAPRGCLAGRVRDACGCCWECANLEGQLCDLDPSAHFYGHCGEQLECRLDTGGDLSRGEVPEPLCACRSQSPLCGSDGHTYSQICRLQEAARARPDANLTVAHPGP C and thepolypeptide comprising the amino acid sequence of:VPEPLCACRSQSPLCGSDGHTYSQICRLQEAARARPDANLTVAHPGPC.

The protein of SEQ ID NO:80 (KSPI1) is a 267-amino-acid long protein,and is a new variant of the bA108L7.1 gene (Genbank accession numberAL133215). The 255 first amino-acids are identical between the twoproteins, but the 12 last amino-acids of KSPI1 are unique. KSPI1displays a signal peptide (MLPPPRPAAALALPVLLLLLVVLTPPPTGA), a kazal-typeserine protease inhibitor (Ki) domain

(VPEPLCACRSQSPLCGSDGHTYSQICRLQEAARARPDANLTVAHPGPC), anImmunoglobulin-like (Ig) domain

(QDVIFGCEVFAYPMASIEWRKDGLDIQLPGDDPHISVQFRGGPQRFEVTGWLQIQAVRPS DEGTYRCLG)and an Insulin-like growth factor-binding domain

(CAPCRPEECAAPRGCLAGRVRDACGCCWECANLEGQLC). Furthermore, KSPI1 displayshomologies with many Insulin-like growth factor-binding proteins (IGFBP)from positions 1 to 255, and highest homology with a well-known IGFBP isobtained with human MAC25.

KSPI1 is a new Kazal-type serine protease inhibitor. Protease inhibitorsare important tools of nature for regulating the proteolytic activity oftheir target proteases, for blocking these in emergency cases, or forsignaling receptor interaction or clearance. Kazal-type serine proteasesinhibitors have been shown to inhibit a number of serine proteases suchas trypsin, elastase, acrosin, and thrombin. As these proteases areinvolved in major biological processes such as haemostasis, inflammationand apoptosis, their inhibitors may have a wide range of therapeuticalapplications (Dahlback, Lancet, 355:1627-32 (2000); Watorek et al., AdvExp Med Biol, 240:23-31 (1988); Martin et al, Cell, 82:349-52 (1995)).

Moreover, KSPI1 belongs to the low affinity IGFBP family. IGFBPs aresoluble proteins that bind insulin-like growth factors (IGFs). IGFs areinvolved in the regulation of cellular growth and metabolism, and theprincipal function of IGFBPs is to regulate IGF availability in bodyfluids. Some serine protease inhibitors have been shown to be implicatedin the activation of growth factors (Kawaguchi et al, J Biol Chem272:27558-64 (1997)). As KSPI1 is a serine protease that belongs to thelow affinity IGFBP family, KSP1 binds to IGFs and modulates IGFactivation by inhibiting a serine protease.

An embodiment of the present invention relates to methods of using KSPI1or fragment thereof to inhibit contaminating proteases in a sample. Inparticular, KSPI1 can be used in a “cocktail” of protease inhibitorsthat is able to inhibit a wide range of proteases without knowing thespecificity of any of the proteases. Such protease inhibitor cocktailsare widely used in laboratory assays to prevent degradation of proteinsamples by contaminating proteases.

In another embodiment, KSPI1 or fragment thereof can be used to treatand/or attenuate thrombin-mediated and thrombin-associated diseases.Thrombin is a serine protease that regulates the last step in thecoagulation cascade, and has a central regulatory role in haemostasisand thrombus formation. Any compositions and methods containing, e.g.,KSPI1 or fragment thereof, a polynucleotide encoding the protein, or acompound that increases the expression or activity of KSPI1. Suchcompounds can be readily identified, e.g. by screening test agentsagainst cells expressing KSPI1 and detecting the ability of the testagents to increase the level of KSPI1 expression. A method fordetermining the ability of the polypeptides of the invention to blockthe proteolytic activity of thrombin is described in U. S. Pat. No.6,218,365. The compositions of the invention can be administereddirectly to the patient using any suitable method, for example byintravenous perfusion or by oral administration. The compositions of theinvention can also be used in extracorporeal circuits, as necessary indialysis and surgery. Effective doses of the polypeptides of the presentinvention are determined according to the relevant techniques. Thecompositions of the invention may be administered alone or incombination with other known agents inhibiting proteases of thecoagulation cascade. Thrombin-mediated and thrombin-associated diseasesin which the coagulation cascade is activated include but are notlimited to deep vein thrombosis, pulmonary embolism, thrombophlebitis,arterial occlusion from thrombosis or embolism, arterial reocclusionduring or after angioplasty or thrombolysis, restenosis followingarterial injury or invasive cardiological procedures, postoperativevenous thrombosis or embolism, acute or chronic atherosclerosis, stroke,myocardial infarction.

In another preferred embodiment, KSPI1 or fragment thereof can be usedto treat and/or attenuate diseases associated with neutrophil-releasedproteases. Activated neutrophils release serine proteases such aselastase or cathepsin G, which result in abnormal connective tissueturnover and in severe damage to healthy tissues if not properlycontrolled [Watorek et al., Adv Exp Med Biol, 240:23-31 (1988)]. KSPI1or fragment thereof inhibit neutrophil-released proteases, andinhibition efficiency of the polypeptides of the invention can bedetermined by measuring in vitro the apparent equilibrium dissociationconstants using methods derived for tight-binding inhibitors [Bieth,Proteinase Inhibitors. 463-9 (1974); Williams et al, Methods Enzymol.63:437-67 (1979)]. The compositions of the invention can be administereddirectly to the patient using any suitable method, for example byintravenous perfusion or by oral administration. Effective doses of thepolypeptides of the present invention are determined according to therelevant techniques. The compositions of the invention may beadministered alone or in combination with other known agents inhibitingneutrophil-released proteases. Diseases caused by neutrophil-releasedproteases include but are not limited to emphysema, idiopathic pulmonaryfibrosis, adult respiratory distress syndrome, cystic fibrosis,rheumatoid arthritis, organ failure, glomerulonepritis and variousinflammatory diseases.

Another embodiment of the invention relates to the inhibition and/orattenuation of proteases produced by pathogenic microorganisms. Thisembodiment relates to the administration of KSPI1 or fragment thereof,or a compound that increases the expression or activity of KSPI1, aloneor in combination with other known agents, for preventing and/ortreating parasitic infections in human, in animals and in cell cultures.It has previously been shown that protease inhibitors can preventdissemination of a virus, a protozoa, a bacteria or a fungus in the hostorganism. Methods for determining the ability of the polypeptides of theinvention to block the proteolytic activity of serine proteases fromvarious pathogenic microorganisms, for preparing and evaluating thepharmaceutical compositions, and for administrating the compositions aredescribed in U.S. Pat. No. 5,739,283, which disclosure is herebyincorporated in its entirety. Accordingly, the polypeptides of thepresent invention may be used to prevent or to treat, e.g., coccidiosis,staphylococcal infection, infection by the influenza virus, P.gingivalis or T. denticola, and invasive pulmonary aspergillosis.

Another embodiment of the present invention relates to methods of usingKSPI1 or fragment thereof to remove or to purify serine proteases in asample. Such methods can be useful either for removing contaminatingproteases from a sample or for purifying a given protease in a sample.Preferred polypeptides are KSPI1 in its entirety, polypeptidescontaining the Ki domain, and polypeptides containing the Ki and the Igdomains. Recombinant proteins that display the Ki and/or Ig of KSPI1 mayalso be used. The binding efficacity of KSPI1 to a given serine proteasecan be tested using any suitable method, e.g., immunoprecipitation andWestern blots analysis. Any method of binding KSPI1 to the protease andof purifying the complex can be used in such methods. In a preferredembodiment, KSPI1 or fragment thereof may be bound to a chromatographicsupport, either alone or in combination with other proteases inhibitors,to form an affinity chromatography column. The sample to analyse couldthen be run through this affinity chromatography column.

Another embodiment of the present invention relates to methods of usingKSPI1 or fragment thereof to detect and/or to quantify the amount ofprotease in a sample, and thus to use these methods in assays anddiagnostic kits for the quantification of proteases in samples, bodilyfluids or cell cultures. Such assays can be used to calculate the yieldof a serine protease purification, and such diagnosis kits, which alsocontain a sample representative of the amount of protease found in anormal subject, can be used to detect diseases and disorders caused as aresult of protease activity, including those listed above. Preferredpolypeptides are KSPI1 in its entirety, polypeptides containing the Kidomain, and polypeptides containing the Ki and the Ig domains. Anymethod of detecting the protease inhibitor activity of KSPI1 or fragmentthereof can be used in such methods. For example, the sample is assayedusing a standard protease substrate. A known concentration of KSPI1 orfragment thereof is added, and allowed to bind to a particular proteasepresent. The protease assay is then rerun, and the loss of activity isthen correlated to the protease inhibitor activity using techniqueswell-known to those skilled in the art.

Still another embodiment of the invention relates to compositions andmethods for modulating IGF activity by decreasing binding of KSPI1 toIGFs. Compounds that inhibit the interaction of an IGF with any one ofits binding proteins and not to a human IGF receptor are useful toincrease serum and tissue levels of active IGFs in a mammal. Thus,compositions and methods for decreasing binding of KSPI1 to IGFs can beused, for example, in any treatments where IGFs are usuallyadministrated, e.g., treatment of hyperglycemic, obesity-related,neurological, cardiac, renal, immunologic, and anabolic disorders. Theinhibition and/or reduction of binding of KSPI1 to IGFs can be achievedusing any suitable method, e.g. through the administration of atherapeutically effective amount of an antibody that specificallyrecognizes KSPI1 or fragment thereof to a patient. Preferably, twoantibodies are used, separately or simultaneously, one recognizing theIg domain and the other recognizing the Ki domain. The antibody can beadministered alone or in combination with one or more agent known in theart, e.g. those described in U.S. Pat. No. 6,251,865, which disclosureis hereby incorporated by reference in its entirety. Effective doses ofthe antibodies of the present invention are determined according to therelevant techniques. Decreased binding of KSPI1 to IGFs can also beobtained by using methods and compounds decreasing KSPI1 expression oractivity. Such methods and compounds include, but are not limited to,antisense molecules, ribozymes, dominant negative forms of KSPI1, andcompounds that decrease the activity or expression of KSPI1 in a cell.

Another embodiment of the present invention relates to methods of usingKSPI1 or fragment thereof to purify IGFs in a sample. Such methods ofpurifying IGFs can be used to analyse the different IGFs present in apatient suffering of one of the diseases listed above. The bindingefficacity of KSPI1 to a given IGF can be tested using any suitablemethod, e.g., immunoprecipitation and Western blots analysis. Any methodof binding KSPI1 to IGFs and of purifying the complex can be used insuch methods. In a preferred embodiment, KSPI1 or fragment thereof maybe bound to a chromatographic support, either alone or in combinationwith other IGFBPs, to form an affinity chromatography column. The sampleto purify could then be run through this affinity chromatography column.In another series of embodiments, KSPI1 or fragment thereof can be usedto detect and/or to quantify IGFs in a sample. Such methods may then beused in assays and diagnostic kits for the quantification of KSPI1-IGFcomplexes in, e.g., bodily fluids or tissue samples. Any method ofdetecting the presence or level of KSPI-IGF complexes can be used. Inparticular, the methods described by Khosravi et al [Clin Chem 43:523-32(1997)] and in U.S. Pat. No. 6,248,546, which disclosures are herebyincorporated by reference in their entirety, may be adapted. In apreferred embodiment, the diagnosis kit, which contains a samplerepresentative of the level of KSPI1-IGF complexes found in a normalsubject, can be used to detect diseases caused as a result of impairedIGF level, or to monitor the effects of a treatment aiming to increaseor decrease IGF level in a patient.

Protein of SEQ ID NOS: 82 (Internal Designation Clone517778_(—)184-5-3-0-G3-F)

The cDNA of Clone 517778_(—)184-5-3-0-G3-F (SEQ ID NO:81) encodes theAmyloid Apoptotic Receptor (AAR) protein comprising the amino acidsequence:

MAGGVRPLRGLRALCRVLLFLSQFCILSGGESTEIPPYVMKCPSNGLCSRLPADCIDCTTNFSCTYGKPVTFDCAVKPSVTCVDQDFKSQKNFIINMTCRFCWQLPETDYECTNSTSCMTVSCPRQRYPANCTVRDHVHCLGNRTFPKMLYCNWTGGYKWSTALALSITLGGFGADRFYLGQWREGLGKLFSFGGLGIWTLIDVLLIGVGYVGPADGSLYI (SEQ ID NO:82). Accordingly, itwill be appreciated that all characteristics and uses of thepolypeptides of SEQ ID NO:82 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in Clone 517778_(—)184-5-3-0-G3-F. In addition, it willbe appreciated that all characteristics and uses of the polynucleotidesof SEQ ID NO:81 described throughout the present application alsopertain to the nucleic acids included in Clone 517778_(—)184-5-3-0-G3-F.A preferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:81, SEQ ID NO:82, and Clone517778_(—)184-5-3-0-G3-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

AAR is a 221 amino acid receptor with two transmembrane segments. Theresulting protein has a hydrophilic intracellular loop and extracellularamino- and carboxy-terminal ends. The transmembrane domains andintracellular loop of AAR are very similar to the third and forthtransmembrane domains and intervening sequence of seven transmembrane Gprotein coupled receptors. The G protein-binding amino acid sequence DRFis found near the first transmembrane region of AAR. The extracellularportion of AAR binds to ligands that include amyloidgenic peptides.Ligand binding leads to apoptosis for the AAR-expressing cell. AAR hasbiological activities that comprise binding G protein components andligands such as amyloidgenic peptides.

Preferred embodiments of the invention include:

A method of preventing cell death wherein a ligand-binding polypeptidefragment of AAR is contacted with ligand in an amount effective tocompetitively inhibit ligand binding to AAR expressed on a cell.

Preferred polypeptide fragments of AAR include but are not limited tothose starting at an amino acid chosen from amino acids 1-40 and endingat amino acid chosen from amino acids 165-180. The most preferredpolypeptide fragment comprises amino acids 1-180 of AAR.

Preferred forms of inhibited cell death include those associated withamyloidgenic peptides. A method of inducing apoptotic cell death whereinan AAR ligand is contacted with a cell in an amount effective to induceapoptosis of the cell. Preferred AAR ligands include amyloidgenicpeptides. Further preferred AAR ligands are compounds that bindspecifically to AAR and cause apoptosis in the cell expressing AAR.Further preferred AAR ligands include AAR-specific antibodies. PreferredAAR-specific antibodies include those that bind an epitope within theamino-terminal extracellular region of AAR. Preferred cells to becontacted with AAR ligand include neoplastic cells. Further preferredcells include neoplastic cells that express AAR.

A preferred solution of AAR ligand further comprises a hydrogel-formingpolymer solution to improve localization of delivery. A preferredsolution of AAR ligand further comprises one or more alkaline salts toimprove ligand binding. A preferred solution of AAR ligand furthercomprises one or more chemotherapeutic agents to improve efficacy oftreatment. A preferred method of contacting a cell includes catheterinjection.

A preferred method of contacting a cell further includes tumor imagingto improve accuracy of localized delivery. A preferred method ofcontacting a cell further includes computer modeling and administrationof AAR ligand to improve accuracy of localized delivery.

The amino-terminus of AAR is capable of binding to ligands such asamyloidgenic peptides (i.e., the β-amyloid peptide associated withAlzheimer's disease, Amyloid Precursor Like Proteins (APLP) 1 and 2,immunoglobulin light chain, prealbumin, β-2-microglobulin,transthyretin, amylin, insulin, atrial natriuretic peptide (ANP),apolipoproteins and glucagon). The amyloidgenic fragments of theseproteins form predominantly beta-pleated sheet structures that may adoptthe fibrillar configuration of amyloid in certain pathologic states.Amyloid deposits often lead to cell death in affected tissues.Amyloid-associated disorders include, most notably, Alzheimer's disease,diabetes, systemic amyloidosis, familial visceral amyloidosis, cutaneousamyloidosis, Muckle-Wells syndrome, Gerstman-Straussler disease,dialysis-related and hemodialysis-related amyloidosis. Amyloid depositsmay lead to further pathogenic outcomes depending on the affectedtissue. For instance, hemodialysis-related amyloidosis can result incarpal tunnel syndrome, erosive arthropathy, spondyloarthropathy, lyticbone lesions, and pathologic fractures. β-amyloid peptide deposition inthe tunica media of leptomeningeal and parenchymal vessels causesdegradation of smooth muscle cells and subsequent cortical hemorrhages.Furthermore, the neuronal cell death observed in Alzheimer's disease isassociated with the senility that accompanies the later stages of thedisease and pancreatic β-islet cell death is a causative factor ofdisrupted insulin regulation in diabetes. Reducing the level ofamyloidgenic peptides is a desired therapy for disorders such as thoselisted herein.

In a preferred embodiment of the invention, a ligand-binding polypeptidefragment of AAR is used to prevent cell death. This method comprises thestep of: contacting a ligand-binding fragment of AAR with ligand in anamount effective to competitively inhibit binding of ligand to AARexpressed on a cell. Preferred polypeptide fragments of AAR include butare not limited to those starting at an amino acid chosen from aminoacids 1-40 and ending at an amino acid chosen from amino acids 165-180.Any single AAR fragment or combination of AAR fragments included in saidlist may be excluded from this embodiment of the invention. The mostpreferred fragment comprises amino acids 1-180 of AAR. Preferred formsof inhibited cell death include those associated with amyloidgenicpeptides, such as pancreatic β-islet cell death and others listedherein. AAR fragments may be applied by methods common to the art suchas those discussed herein. For example, AAR fragments may be deliveredto cells of the pancreas in physiologically acceptable form by directinjection or catheter. For prolonged treatment, AAR fragments may bereleased from an implantable polypeptide-releasing stent (U.S. Pat. Nos.5,683,345 and 5,500,013, which disclosures are hereby incorporated byreference in their entireties).

In the absence of ligand, AAR expression protects a cell from apoptoticcell death. AAR is expressed in many different cell types, includingleukocytes and cells of the heart, brain, placenta, ovaries, testes,lung, liver, muscle, kidney, pancreas, colon, intestine, and prostate.Therefore, AAR may be exploited to cause cell death by addition ofligand. This inducible cell death is useful for treating neoplastic cellgrowth in a number of different tissues. As a preferred embodiment ofthe invention, an AAR ligand is used in a method to promote apoptoticcell death. This method comprises the step of contacting an AAR ligandwith a cell in an amount effective to induce apoptotic cell death.Preferred AAR ligands include but are not limited to those listed herein(i.e., amyloidgenic peptides). Any single amyloidgenic peptide ligand orcombination of amyloidgenic peptide ligands may be excluded from thisembodiment of the invention. Further preferred AAR ligands are compoundsthat bind specifically to AAR and cause apoptosis in the cell expressingAAR, such as an AAR-specific antibody. Preferred antibodies for use inthis method include those that bind an epitope within the amino-terminalextracellular region of AAR. Any single antibody or combination ofantibodies that bind to an epitope of AAR may be excluded from thisembodiment of the invention. Preferred cells to be contacted with AARligand include neoplastic cells including but not limited to: neoplasticleukocytes and neoplastic cells of the heart, brain, placenta, ovaries,testes, lung, liver, muscle, kidney, pancreas, colon, intestine, andprostate. Further preferred cells include those that express AAR.Delivery of AAR ligand to specific cells may be accomplished by methodscommon to the art such as those discussed herein. For example, aneffective amount of AAR ligand in physiologically acceptable solutionmay be injected locally by syringe or catheter into a tumor mass topromote apoptotic cell death. AAR ligand may be used as the sole activeagent in the solution, or may be used in combination with otherchemotherapeutic drugs to increase the efficacy of treatment. A problemwith direct delivery of AAR ligand into a solid tumor may be resistanceof the tissue to the influx of the fluid. Increased penetration and/orreduced backflow through the point of entry, so that more material isintroduced into and remains in the tumor, is obtained through the use ofa viscous vehicle for the AAR ligand. Preferred materials includesolutions or suspensions of a polymeric material which form a hydrogelat the time of or shortly after injection or implantation. The hydrogelsolution of AAR ligand is injected via a catheter into regions of thetumor to be treated as described in U.S. Pat. No. 5,945,100, whichdisclosure is hereby incorporated by reference in its entirety. Anotherproblem with direct delivery of AAR ligand is that cancerous tumorsgenerate localized areas of relatively high acidity due to a metabolicprocess known as “anaerobic glycolysis.” This acidic environment mayinterfere with ligand binding to AAR. A physiologically acceptablesolution of AAR ligand may therefore include a variety of alkalinesalts, as described in U.S. Pat. No. 5,681,857, which disclosure ishereby incorporated by reference in its entirety. To further increasethe accuracy of treatment, tumor imaging, alone or in combination withcomputer modeling and administration of the AAR ligand solution may beemployed (U.S. Pat. Nos. 5,438,989 and 5,823,993, which disclosures arehereby incorporated by reference in their entireties).

Proteins of SEQ ID NOs:84, 86, and 98 (Internal Designation Clones100038_(—)105-017-4-0-E4-F, 100523_(—)105-019-1-0-F3-F, and100545_(—)105-019-2-0-E3-F)

The cDNAs of Clones 100038_(—)105-017-4-0-E4-F and100523_(—)105-019-1-0-F3-F NOs:83 and 85, respectively) encode theSoluble Activator of Wnt (SAW)-l protein comprising the amino acidsequence:

MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQGQCGEGHPARTLPPRPLGQP SRRRFQVPGPS(SEQ ID NOs:84 and 86). The cDNA of Clone 100545_(—)105-019-2-0-E3-F(SEQ ID NO:97) encodes the SAW-2 protein comprising the amino acidsequence:MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQGQCGEGAEVGLLSPCCGTR GEENWFAEVA(SEQ ID NO:98). Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NOs:84, 86, and98 described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in Clones100038_(—)105-0174-0-E4-F, 100523_(—)105-019-1-0-F3-F, and100545_(—)105-019-2-E3-F, respectively. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NOs:83, 85, and 97 described throughout the present applicationalso pertain to the nucleic acids included in Clones100038_(—)105-017-4-0-E4-F, 100523_(—)105-019-1-0-F3-F, and100545_(—)105-019-2-0-E3-F, respectively. A preferred embodiment of theinvention is directed toward the compositions of SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:97, SEQ ID NO:98, Clone100038_(—)105-017-4-0-E4-F, Clone 100523_(—)105-019-1-0-F3-F, and Clone100545_(—)105-019-2-0-E3-F. Also preferred are fragments having abiological activity described herein and the polynucleotides encodingthe fragments. A preferred fragment of the polypeptides of SEQ ID NOs:84and 86 comprises:MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQGQCGEGHPARTLPP. A preferredfragment of the polypeptides of SEQ ID NO:98 comprises:MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQGQCGEGAEVGLLSP. A furtherpreferred fragment of the polypeptide sequences of SEQ ID NOs:84, 86,and 98 comprises:MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQGQ.

A list of preferred embodiments of the invention follows. A preferredembodiment is a composition, comprising a SAW-1 polypeptide sequence ofSEQ ID NO:84.

A preferred embodiment is a composition, comprising a SAW-1 polypeptidesequence of SEQ ID NO:86.

A preferred embodiment is a composition, comprising a SAW-1 polypeptidefragment having biological activity.

A preferred embodiment is a composition, comprising a SAW-2 polypeptidesequence of SEQ ID NO:98.

A preferred embodiment is a composition, comprising a SAW-2 polypeptidefragment having biological activity.

A preferred embodiment is a composition, comprising a polynucleotidesequence of SEQ ID NO:83 encoding a SAW-1 polypeptide.

A preferred embodiment is a composition, comprising a polynucleotidesequence of SEQ ID NO:85 encoding a SAW-1 polypeptide.

A preferred embodiment is a composition, comprising a polynucleotidesequence encoding a biologically active SAW-1 polypeptide fragment.

A preferred embodiment is a composition, comprising a polynucleotidesequence of SEQ ID NO:97 encoding a SAW-2 polypeptide.

A preferred embodiment is a composition, comprising a polynucleotidesequence encoding a biologically active SAW-2 polypeptide fragment.

A preferred embodiment is a method of increasing Wnt-dependent signalingto facilitate stem cell growth comprising the step of: contacting aSAW-1 or SAW-2 polypeptide or biologically active fragment thereof witha stem cell.

Preferred stem cells include those capable of growth or proliferation inresponse to Wnt.

Further preferred stem cells include those capable of giving rise tohematopoetic cells.

Further preferred stem cells include those capable of giving rise toneuronal or neuroglial cells.

Further preferred stem cells include those capable of giving rise tohepatocytes.

Further preferred stem cells include those capable of giving rise topancreatic cells.

Further preferred stem cells include osteoblasts.

Further preferred stem cells include chondroblasts.

Further preferred stern cells include those found in cord blood.

Also preferred is the addition of one or more cell-type specific growthfactor to the stem cell before, during or subsequent to contact withSAW-1 or SAW-2 polypeptide.

A preferred embodiment is a method of increasing Wnt-dependent signalingto prevent apoptosis comprising the step of: contacting a SAW-1 or SAW-2polypeptide or biologically active fragment thereof with a cell at riskof apoptosis.

Preferred cells are those capable of responding to Wnt.

Preferably, the method is applied to prevent apoptosis of cells inculture.

Preferably, the method is applied to treat an apoptosis-relateddisorder.

Preferably, the method is applied to prevent an apoptosis-relateddisorder.

A preferred apoptosis-related disorder is chosen from the listconsisting of: neurodegenerative diseases, Spinal Muscular Atrophy (SMA)types I-III, Amyltrophic Lateral Sclerosis (ALS), Huntington's disease,Alzheimer's disease, Parkinson's disease, retinal degeneration,retinitis pigmentosa, cerebellar degeneration, myelodysplasis, aplasticanemia, ischemia-related degeneration, myocardial infarction, stroke,hepatic degeneration diseases, alcoholic hepatitis, hepatitis B,hepatitis C, fulminant hepatitis, joint degeneration diseases,osteoarthritis, and diabetes.

SAW-1 and SAW-2 are splice variants of the Wnt-6 gene. In the case ofSAW-1, the 135-nucleotide cassette inserted into the Wnt-6 cDNA encodesan early termination codon. The resulting SAW-1 polypeptide is 131 aminoacids in length, compared to the 365-amino acid Wnt-6 protein. In thecase of SAW-2, a 236-nucleotide insertion also encodes for an earlytermination codon. SAW-2 polypeptide is 129 amino acids in length andpossesses a biological activity identical to that of SAW-1. The Wntfamily of proteins is crucial for determining cell polarity and fate,patterning of a number of tissues in the developing embryo, cellproliferation, and maintenance of stem cell populations throughout life.The role of Wnt proteins in promoting cell survival may explain theprevalence of Wnt overexpression in human cancers. Wnt proteins aresecreted factors that generally associate with the extracellular matrixor cell surface. Receptors for Wnt proteins include the Frizzled (Fz)family of seven transmembrane spanning receptors and the low-densitylipoprotein receptor-related proteins (LRP) 5 and 6. These receptors canact synergistically as Wnt coreceptors to transmit signals andupregulate target gene expression. Inhibitors of Wnt signaling include asoluble form of the Fz receptor, which acts as a competitive dominantnegative inhibitor, and the extracellular factors Cerberus andWnt-Inhibitory Factors (WIFs). Therefore, Wnt proteins are targets formultiple protein-protein interactions. SAW-1 and SAW-2 are novel,truncated splice variants of Wnt-6 that interact with Cerberus and WIFproteins. The biological activities of SAW-1 and SAW-2 are defined bythose interactions.

Wnt proteins are important in maintaining stem cell populationsthroughout adulthood. Stem cells comprise an undifferentiated orpartially undifferentiated self-renewing population. As used herein,“stem cell” refers to any cell that retains undifferentiated character,is capable of self-renewal, and that gives rise to a furtherdifferentiated cell. These cells are important for renewing cellpopulations of nearly every type, especially the high-turnoverpopulations of epithelial linings, dermal layers, and the reproductiveand hematopoetic systems. Defects in stem cell populations or drasticcell loss, whether caused by genetic predisposition, trauma, injury,disease, or medical treatments such as chemotherapy, have a disastrouseffect on an individual. These defects may be overcome by stimulatinggrowth of the remaining stem cell population in vivo. Alternatively, invitro culture and transplantation of stem cells, preferably derived fromthe individual in need of treatment, but also from other sources such ascord blood, may be effective. Mature cells derived from the culturedstem cells may be transplanted as well. As Wnt proteins are effectivegrowth and survival factors for stem cells, these proteins are usefulfor either strategy of cell replacement. However, Wnt proteins aredifficult to purify in soluble form and do not diffuse readily, makingWnt-based treatments difficult to execute. A preferred method ofincreasing Wnt signaling is to decrease interaction of Wnt with solubleinhibitors such as Cerberus and WIF.

In a preferred embodiment of the invention, a SAW-1 or SAW-2 polypeptideor biologically active fragment thereof is used to increaseWnt-dependent signaling and facilitate stem cell growth. This methodcomprises the step of contacting a SAW-1 or SAW-2 polypeptide orbiologically active fragment thereof with a stem cell. Preferred stemcells include those capable of growth or proliferation in response toWnt. Also preferred is the addition of one or more cell-type specificgrowth factors or cytokines in combination with SAW-1 or SAW-2polypeptide. Examples include the interleukins (e.g., IL-3),granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(G-CSF), erythropoietin (Epo), lymphotoxin, steel factor (SLF), tumornecrosis factor (TNF) and gamma-interferon. IL-3 acts on multipotentstem cells as well as progenitors restricted to the granulocyte,macrophage, eosinophil, megakaryocyte, erythroid or mast cell lineages,while Epo acts on fairly mature erythroid progenitor cells. SAW-1 orSAW-2 polypeptide or a biologically active fragment thereof may be usedto facilitate stem cell proliferation in culture by adding aphysiologically acceptable solution comprising said polypeptide to astem cell in culture (e.g., liver stem cells, neural or neuroglial stemcells, osteoblasts, chondroblasts, pancreatic stem cells, hematopoeticstem cells, cord blood, etc.) in an amount effective to promoteWnt-dependent growth or proliferation. A physiologically acceptablesolution comprising a SAW-1 or SAW-2 polypeptide or a biologicallyactive fragment thereof may further be added upon transplantation orreintroduction of cultured cells into an individual to provideadditional growth potential for the cells in vivo. Cell transplantationand reintroduction methods are determined by one skilled in the art andinclude injection of a single-cell suspension by syringe or catheter andsurgical implantation (also see U.S. Pat. No. 5,869,463 for neuroglialcell transplants; U.S. Pat. No. 6,068,836 for bone marrow transplants;Noel et al., Metabolism, 31:184-7 (1982) for pancreatic celltransplants; and U.S. Pat. Nos. 4,950,296, 5,385,566, and 6,200,324 forbone transplants, which disclosures are hereby incorporated by referencein their entireties). Additionally this method may be applied toincrease Wnt-dependent stem cell growth and proliferation in vivo. Forexample, a physiologically acceptable solution comprising SAW-1 or SAW-2polypeptide or a biologically active fragment thereof may be directlyinjected to the site of interest (e.g., the bone marrow for hematopoeticstem cell treatment) or by other methods common to the art. Given thatstem cells are, at the earliest stage, able to differentiate into almostany kind of mature, functional cell, a wide variety of conditions may beaddressed by stem cell treatment. As an example, hematopoetic stem cellgrowth or replacement may benefit those predisposed to or sufferingfrom, any one or more of the following exemplary conditions:lymphocytopenia; lymphorrhea; lymphostasis; immunodeficiency (e.g., HIVand AIDS); infections (including, for example, opportunistic infectionsand tuberculosis (TB)); lupus; disorders characterized by lymphocytedeficiency, erythrocytopenia; erthrodegenerative disorders;erythroblastopenia; leukoerythroblastosis; erythroclasis; thalassemia;anemia (e.g., hemolytic anemia, such as acquired, autoimmune, ormicroangiopathichemolytic anemia; aplastic anemia; congenital anemia,e.g., congenital dyserythropoietic anemia, congenital hemolytic anemiaor congenital hypoplastic anemia; dyshemopoietic anemia; Faconi'sanemia; genetic anemia; hemorrhagic anemia; hyperchromic or hypochromicanemia; nutritional, hypoferric, or iron deficiency anemia; hypoplasticanemia; infectious anemia; lead anemia; local anemia; macrocytic ormicrocytic anemia; malignant or pernicious anemia; megaloblastic anemia;molecular anemia; normocytic anemia; physiologic anemia; traumatic orposthemorrhagic anemia; refractory anemia; radiation anemia; sickle cellanemia; splenic anemia; and toxic anemia); myelofibrosis;thrombocytopenia; hypoplasia; disseminated intravascularcoagulation(DIC); immune (autoimmune) thrombocytopenio purpura (ITP); HIV inductedITP; myelodysplasia; thrombocytotic diseases and thrombocytosis. Stemcells giving rise to neural or neuroglial cells may be applied to treatdisorders including but not limited to: Alzheimer's disease,frontotemporal dementia, bipolar disorder, Huntington's chorea, multiplesclerosis, amyotrophic lateral sclerosis, Tay-Sach's disease, Gaucher'sdisease, and dopamine-related disorders such as Parkinson's disease andschizophrenia. Pancreatic stem cell cultures may be applied to treatmentof metabolic disorders such as diabetes. Stem cells from bone tissue(osteoblasts) may be used to treat to bone loss, atrophy, ormalformation due to injury, congenital or chronic conditions,osteopenia, osteoporosis, rickets, malignant melanoma-induced bonedegradation, and bone fissures or fractures due to injury, electivesurgery (e.g., plastic surgery), reconstructive surgery, and dentalprocedures or surgeries.

Wnt proteins act to inhibit apoptosis and promote survival ofWnt-responsive cells. A specific activator of Wnt signaling is desirableboth for cell or tissue growth in vitro and for treatingapoptosis-related disorders in vivo. Examples of such disorders include:neurodegenerative diseases such as Spinal Muscular Atrophy (SMA) typesI-III, Amyltrophic Lateral Sclerosis (ALS), Alzheimer's disease,Huntington's disease, Parkinson's disease, retinal degeneration,retinitis pigmentosa and cerebellar degeneration; myelodysplasis such asaplastic anemia; ischemic diseases such as myocardial infarction andstroke; hepatic diseases such as alcoholic hepatitis, hepatitis B,hepatitis C, and fulminant hepatitis; joint-diseases such asosteoarthritis; and metabolic disorders such as diabetes. In a preferredembodiment of the invention, a SAW-1 or SAW-2 polypeptide orbiologically active fragment thereof is used to preventapoptosis-related degeneration. This method comprises the step ofcontacting a SAW-1 or SAW-2 polypeptide or biologically active fragmentthereof with a cell. Preferred cells are those capable of responding toWnt. Further preferred cells are those at risk of apoptosis. Forexample, a physiologically acceptable composition comprising SAW-1 orSAW-2 polypeptides may be added to a mixed culture of hippocampalneurons to improve cell survival in culture. Alternatively, aphysiologically acceptable composition comprising SAW-1 or SAW-2polypeptide or biologically active fragment thereof may be delivered toan individual diagnosed with or at risk of an apoptosis-relateddisorder, as determined by one skilled in the art. SAW-1 or SAW-2polypeptide may be used alone or in combination with agents thatmodulate Wnt signaling, apoptosis, or cell type-specific processes.Furthermore, SAW-1 or SAW-2 polypeptide may be fused to a ligand for thepurpose of stabilizing and/or targeting said polypeptide (for example,tetanus toxin, calcium channel blocking agents, transferrin, poliovirusepitopes, neuropeptide fragments, or steroid hormone androgens, orfragments thereof which are sufficient for neuronal targeting). As anexample, a physiologically acceptable composition comprising SAW-1 orSAW-2 polypeptides may be delivered to an individual to preventosteoarthritis-associated joint degeneration. As an additional example,said composition may be administered to an individual that has or islikely to experience an ischemic event. Appropriate delivery methods,such as those discussed herein, may be determined on a case by casebasis by one skilled in the art.

Protein of SEQ ID NOs:88 and 90 (Internal Designation Clone116470_(—)105-063-3-0-H7-F and Clone 122600_(—)105-077-3-0-F9-F)

The cDNAs of Clone 116470_(—)105-063-3-0-H7-F and Clone122600_(—)105-077-3-0-F9-F (SEQ ID NOs:87 and 89, respectively) encodethe Dopamine AMPhetamine INhibitor (Dampin) protein comprising the aminoacid sequence:

MLFRLSEHSSPEEEASPHQRASGEGHHLKSKRPNPCAYTPPSLKAVQRIAESHLQSISNLNENQASEEEDELGELRELGYPREEDEEEEEDDEEEEEEEDSQAEVLKVIRQSAGQKTTCGQGLEGPWERPPPLDESERDGGSEDQVEDPALSEPGEEPQRPSPSEPGT (SEQ ID NOs:88 and 90).Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NOs:88 and 90 described throughout thepresent application also pertain to the polypeptides encoded by thenucleic acids included in Clone 116470_(—)105-063-3-0-H7-F and Clone122600_(—)105-077-3-0-F9-F, respectively. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NOs:87 and 89 described throughout the present application alsopertain to the nucleic acids included in Clone116470_(—)105-063-3-0-H7-F and Clone 122600_(—)105-077-3-0-F9-F,respectively. A preferred embodiment of the invention is directed towardthe compositions of SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, Clone 116470_(—)105-063-3-0-H7-F and Clone122600_(—)105-077-3-0-F9F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

Dampin is a splice variant of the Dopamine and cAMP-RegulatedPhosphoProtein-32 (DARPP-32) that utilizes a different translation startsite and lacks the first 37 amino acids of DARPP-32. DARPP-32 is acytoplasmic signaling molecule that is regulated by phosphorylation atresidues T34 by Protein Kinase A (PKA) to function as an inhibitor ofProtein Phosphatase 1 (PP1). This increases the effect of PKA ondownstream targets. In neurons, PKA phosphorylates DARPP-32 in responseto dopamine or psychoactive drugs that act on dopamine signalingpathways (e.g., cocaine and amphetamines). Alternatively,phosphorylation of T75 by Cdk5 results in DARPP-32 inhibition of PKA.Dampin, as a splice variant, is not phosphorylated in response to PKAsignaling and does not act as an inhibitor of PP1. However, Dampin has aCdk5 phosphorylation site and is able to inhibit PKA signaling.

Abnormal signaling through dopaminergic pathways has been implicated inseveral major neurological and psychiatric disorders, includingParkinson's disease, Tourette's syndrome, Attention Deficit Disorder(ADD), Huntington's disease, schizophrenia, and drug/alcohol abuse. Inparticular, cocaine and amphetamines activate the dopaminergic pathwaysthrough PKA. Furthermore, addictive behavior is associated withincreased dopaminergic signaling and PKA activity. Therefore, diminishedPKA activity may be desired to address addictive behavior and drug andalcohol abuse. Increases in dopamine responses may be desired to treatdisorders such as Parkinson's disease, Tourette's syndrome, ADD,Huntington's disease, and schizophrenia.

Progesterone, similar to dopamine, also activates PKA, which leads toDARPP-32 phosphorylation at T34 and inhibition of PP1. Dampin inhibitsboth dopamine and progesterone signaling by attenuating PKA activity.Progesterone is required for ovulation and implantation of a fertilizedegg in the uterine wall. In addition, progesterone, in combination withdopamine increases female sexual receptivity. Therefore, high levels ofDampin relative to DARPP-32 would be effective for female birth controlas well as behavioral modification (e.g., for purposes of animaltraining). Alternatively, high levels of DARPP-32 relative to Dampinwould be effective for increasing female fertility and sexualreceptivity.

PP1 activates glycogen synthase in response to insulin. Glycogensynthesis is one mechanism by which blood glucose levels are regulatedby insulin. DARPP-32 inhibition of PP1 is in turn inhibited by insulin.However, insufficient insulin or insulin resistance may lead toinappropriate inhibition of PP1 and dysregulation of blood glucoselevels. Such dysregulation may result from disorders that include:Noninsulin dependent diabetes mellitus (MIDDM), Insulin dependentdiabetes mellitus (IDDM), insulin resistance and insulin resistantdisorders such as acanthosis nigricans, leprechaunism, and lipoatropahy.As Dampin does not inhibit PP1, high levels of Dampin relative toDARPP-32 would be effective for modulating blood glucose levels byincreasing glycogen synthase activity.

Preferred embodiments of the invention include:

A composition comprising a Dampin polypeptide sequence of SEQ ID NOs:88and 90. A composition comprising a Dampin polypeptide fragment havingbiological activity. A composition comprising a polynucleotide sequenceof SEQ ID NOs:87 and 89 encoding a Dampin polypeptide. A compositioncomprising a polynucleotide sequence encoding a Dampin polypeptidefragment having biological activity.

A method of screening test substances for modulators of Dampinexpression comprising the steps of: i) contacting a cell with a testsubstance; and ii) comparing Dampin expression in the cell afterexposure to the test substance to that of an unexposed control cell.

A method of screening for test substances that modify the ratio ofDARPP-32 relative to Dampin comprising the steps of: i) contacting acell with a test substance; ii) comparing Dampin expression in the cellafter exposure to the test substance to that of an unexposed controlcell; iii) comparing DARPP-32 expression in the cell after exposure tothat of an unexposed control cell; iv) quantifying said expressionlevels; and v) determining the level of DARPP-32 relative to Dampin inthe exposed and unexposed cells.

Preferably, the test substance modifies the ratio of Dampin relative toDARPP-32 in a specific cell type while not in others. Furtherpreferably, the test substance is conjugated to a cell type-specificligand. Preferably, the method screens for test substances that decreasethe ratio of DARPP-32 relative to Dampin. Alternatively, the methodscreens for test substances that increase the ratio of DARPP-32 relativeto Dampin.

A method of differentiating Dampin polypeptides from DARPP-32polypeptides comprising the steps of: i) contacting a first antibodythat binds specifically to DARPP-32 and not Dampin with a proteinsample; ii) contacting a second antibody that binds specifically to bothDARPP-32 and Dampin with a protein sample; and iii) detectingprotein-bound antibody. Preferably, the first and second antibodies arelabeled with a different detectable conjugate. Preferably, the methodfollows immunohistochemical protocols.

A substance that decreases the ratio of DARPP-32 relative to Dampin madeby the process comprising the steps of: i) contacting a cell with a testsubstance; ii) comparing Dampin expression in the cell after exposure tothe test substance to that of an unexposed control cell; iii) comparingDARPP-32 expression in the cell after exposure to that of an unexposedcontrol cell; iv) quantifying said expression levels; v) determining thelevel of DARPP-32 relative to Dampin in the exposed and unexposed cells.

Preferably, the substance decreases the ratio of DARPP-32 relative toDampin in a specific cell type while not in others. Further preferably,the substance is contained in a physiologically acceptable composition.

A substance that increases the ratio of DARPP-32 relative to Dampin madeby the process comprising the steps of: i) contacting a cell with a testsubstance; ii) comparing Dampin expression in the cell after exposure tothe test substance to that of an unexposed control cell; iii) comparingDARPP-32 expression in the cell after exposure to that of an unexposedcontrol cell; iv) quantifying said expression levels; v) determining thelevel of DARPP-32 relative to Dampin in the exposed and unexposed cells.

Preferably, the substance increases the ratio of DARPP-32 relative toDampin in a specific cell type while not in others. Further preferably,the substance is contained in a physiologically acceptable composition.

A method of screening for test substances that specifically bind toDampin and prevent binding to PKA comprising the steps of: i) contactinga test substance with Dampin polypeptide in the presence of PKA, underconditions that allow binding of Dampin to PKA and ii) detecting theamount of PKA bound to Dampin in the presence and absence of the testsubstance.

Preferably, the test substance is able to inhibit Dampin interactionwith PKA in a certain cell type and not in others. Further preferableare test substances conjugated to cell-type specific ligands or portionsthereof.

A substance that specifically binds to Dampin and prevents binding toPKA made by the process comprising the steps of: i) contacting a testsubstance with Dampin polypeptide in the presence of PKA, underconditions that allow binding of Dampin to PKA and ii) detecting theamount of PKA bound to Dampin in the presence and absence of the testsubstance by methods common to the art.

A method of decreasing PKA activity in a neuron comprising the step ofcontacting a substance capable of increasing the ratio of Dampin toDARPP-32 with a neuron. Preferably, the substance is capable of passingthrough the blood brain barrier. Preferably, this method is used todecrease cocaine- or amphetamine-dependent responses. Preferably, thismethod is used to diminish addictive behavior. Further preferably, thismethod is used to diminish alcohol addiction.

A method of decreasing PKA activity in a cell of the female reproductivetract comprising the step of contacting a substance capable ofincreasing the ratio of Dampin to DARPP-32 with a cell of the femalereproductive tract. Preferred cells include ovarian granulosa cells andluteal cells of the uterus. Preferably, this method is used to inhibitprogesterone-dependent ovulation and implantation of a fertilized egg.Preferably, this method is used for female birth control.

A method of modulating blood glucose levels comprising the step ofcontacting a substance capable of increasing the ratio of Dampin toDARPP-32 with a glycogen-storing cell. Preferred glycogen-storing cellsinclude myocytes and hepatocytes.

A method of inhibiting PKA activity comprising the step of introducing aDampin polypeptide into a cell. Preferably, Dampin polypeptide isdelivered to a cell by introducing a polynucleotide encoding Dampinpolypeptide into the cell. Preferably, the polynucleotide is apolynucleotide construct comprising an expression control unit and apolynucleotide encoding Dampin polypeptide. Preferred cells includeneurons, ovarian granulosa cells, uterine cells, hepatocytes, andmyocytes.

A method of increasing neuronal PKA activity comprising the step ofcontacting a substance capable of decreasing the ratio of Dampin toDARPP-32 with a neuron. Preferably, the substance is capable of passingthrough the blood brain barrier. Preferably, this method is used toincrease PKA activity in dopaminergic neurons affected by neurologicaldisorders. Preferred neurological disorders include: Parkinson'sdisease, Huntington's disease, ADD, Tourette's syndrome, andschizophrenia. Preferably, this method is used to increase PKA activityin hypothalamic neurons that express both dopaminergic and progesteronereceptors. Preferably, increasing PKA activity in the hypothalamus isdirected toward increasing sexual receptivity in a female individual.

A method of increasing Atrial Natriuretic Factor (ANF) activitycomprising the step of contacting a substance capable of decreasing theratio of Dampin to DARPP-32 with a nephronic kidney cell. Preferably,this method is used to reduce blood volume. Further preferably, thismethod is used to reduce hypertension.

An embodiment of the invention provides for a method of screening testsubstances for modulators of Dampin expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing Dampin expression in the cell after exposure to the testsubstance to that of an unexposed control cell. Dampin expression isdetermined by methods common to the art or included herein, by detectingDampin polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of Dampin mRNA in eachsample by Northern blot, RTPCR, or another method common to the art. Theinvention provides for design and use of specific polynucleotide probesand primers, as discussed herein. An additional example comprises thesteps of: i) having two equivalent cultures of cells; ii) adding a testsubstance to one of the cultures and not the other; iii) harvesting bothcultures; iv) purifying the protein from each sample of cells; v)comparing the level of Dampin polypeptides in each sample by Westernblot, immunohistochemistry, or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein.

A preferred embodiment of the invention provides a method of screeningfor test substances that modify the ratio of DARPP-32 relative toDampin. This method comprises the steps of: i) contacting a cell with atest substance; ii) comparing Dampin expression in the cell afterexposure to the test substance to that of an unexposed control cell;iii) comparing DARPP-32 expression in the cell after exposure to thetest substance to that of an unexposed control cell; iv) quantifyingsaid expression levels; and v) determining the level of DARPP-32relative to Dampin in the exposed (i.e., test) and unexposed (i.e.,control) cells.

A further preferred embodiment of the invention provides a method ofscreening for test substances that modify the ratio of DARPP-32 relativeto Dampin in a specific cell type while not in others. Included in thismethod are test substances that are conjugated to cell-type specificligands or portions thereof. For example, a test substance may beconjugated to a hydrophilic neuropeptide (e.g., interferon alpha,endorphin, somatostatin) for targeting to the brain (U.S. Pat. No.4,902,505, which disclosure is hereby incorporated by reference in itsentirety).

A preferred embodiment of the invention provides a method of screeningfor test substances that decrease the ratio of DARPP-32 relative toDampin. An alternative preferred embodiment of the invention provides amethod of screening for test substances that increase the ratio ofDARPP-32 relative to Dampin.

In a preferred embodiment of the invention, a substance that decreasesthe ratio of DARPP-32 relative to Dampin is made by the processcomprising the steps of: i) contacting a cell with a test substance; ii)comparing Dampin expression in the cell after exposure to the testsubstance to that of an unexposed control cell; iii) comparing DARPP-32expression in the cell after exposure to that of an unexposed controlcell; iv) quantifying said expression levels; v) determining the levelof DARPP-32 relative to Dampin in the exposed and unexposed cells. Thissubstance is used for purposes discussed herein.

In a preferred embodiment of the invention, a substance that increasesthe ratio of DARPP-32 relative to Dampin is made by the processcomprising the steps of: i) contacting a cell with a test substance; ii)comparing Dampin expression in the cell after exposure to the testsubstance to that of an unexposed control cell; iii) comparing DARPP-32expression in the cell after exposure to that of an unexposed controlcell; iv) quantifying said expression levels; v) determining the levelof DARPP-32 relative to Dampin in the exposed and unexposed cells. Thesubstance that increases the relative level of DARPP-32 is used forpurposes discussed herein.

Methods of detecting Dampin polynucleotides and polypeptides may be usedto detect DARPP-32 polynucleotides and polypeptides and are addressedherein (e.g., mRNA detection methods, antibody-based detection methods).A simple method for differentiating between the Dampin and DARPP-32splice variants is desirable. A preferred embodiment of the inventionprovides a method for differentiating Dampin polypeptides from DARPP-32polypeptides. This method comprises the steps of: i) contacting a firstantibody that binds specifically to DARPP-32 and not Dampin with aprotein sample; ii) contacting a second antibody that binds specificallyto both DARPP-32 and Dampin with a protein sample; and iii) detectingprotein-bound antibody. Preferably, the first and second antibodies arelabeled with a different detectable conjugate. This allows the method tobe carried out with a single protein sample. Preferably, the proteinsample is a fixed, semi-permeablized cell sample. Preferably, thedetection method follows immunohistochemical protocols, as discussedherein.

Dampin inhibits PKA by a competitive binding mechanism. Therefore, theinhibitory effect of Dampin may be ablated by a substance that blocksthe interaction of Dampin with PKA. A preferred embodiment of theinvention provides a method of screening for test substances thatspecifically bind to Dampin and prevent binding to PKA. This methodcomprises the steps of: i) contacting a test substance with Dampinpolypeptide in the presence of PKA, under conditions that allow bindingof Dampin to PKA (e.g., an intact cell); and ii) detecting the amount ofPKA bound to Dampin in the presence and absence of the test substance bymethods common to the art (e.g., antibody-based methods such ascommunopreciptation and Western blotting). Preferably, the testsubstance is able to inhibit Dampin interaction with PKA in a certaincell type and not in others. Included in this method are test substancesthat are conjugated to cell-type specific ligands or portions thereof.

In a preferred embodiment of the invention, a substance that inhibitsDampin binding to PKA is made by the process comprising the steps of: i)contacting a test substance with Dampin polypeptide in the presence ofPKA, under conditions that allow binding of Dampin to PKA (e.g., abiological solution, preferably an intact cell); and ii) detecting theamount of PKA bound to Dampin in the presence and absence of the testsubstance by methods common to the art (e.g., antibody-based methodssuch as communopreciptation and Western blotting).

In a preferred embodiment of the invention, a substance capable ofincreasing the ratio of Dampin to DARPP-32 is used in a method todecrease PKA activity in a neuron. Preferred substances are additionallycapable of passing through the blood brain barrier in vivo. This methodcomprises the step of contacting a neuron with said substance.Diminished activity can be measured by an altered modulation of calciumchannel function in response to dopamine, in situ. This diminishedactivity may also to be measured as a loss of dopamine-mediatedinhibition of the sodium-potassium ATPase (Na,K ATPase) in situ or anincreased excitability of striatal and cortical neurons. This method mayalso be applied to: i) diminish release of dopamine in response toamphetamines, as determined in situ; ii) diminish release of GABA(4-Aminobutyric acid) in response to amphetamines, as determined insitu; iii) increase levels of substance P in the striatum and cortex, asdetermined in situ; iv) increase levels of neurotensin in the striatumand cortex, as determined in situ; v) attenuate increase in locomotoractivity of an individual in response to cocaine; vi) attenuate increasein the protein Fos in response to an amphetamine, as determined in situ;vii) attenuate increase in the protein Chronic Fos Related Antigen (FRA)in response to cocaine, as determined in situ; and viii) diminishinhibition of the activity of the brain sodium-potassium-ATPase inresponse to dopamine, as determined in situ; and ix) decrease addictivebehavior in an individual at risk of or displaying such behavior, asdetermined by family history or clinical assessment.

In a preferred embodiment of the invention, a substance capable ofincreasing the ratio of Dampin to DARPP-32 is used in a method todecrease PKA activity in a cell of the female reproductive tract. Thismethod comprises the step of contacting a cell of the femalereproductive tract with said substance. Preferred cells include ovariangranulosa cells and luteal cells of the uterine tract. Dampin inhibitsprogesterone-mediated PKA activity, which is required for ovulation andimplantation of a fertilized egg in the uterine wall. This method isdirected toward female birth control.

In a preferred embodiment of the invention, a substance capable ofincreasing the ratio of Dampin to DARPP-32 in glycogen-storing cells isused to modulate blood glucose levels. This method comprises the step ofcontacting said substance with glycogen-storing cells. Preferred cellsinclude hepatocytes and myocytes. DARPP-32 inhibits PP1, which isrequired for glycogen synthase activity in response to insulin. Dampindoes not inhibit PP1 and therefore will allow glucose processing andblood glucose modulation. This method is particularly useful formodulating glucose levels in insulin-deficient and diabetic individuals.

As Dampin acts as a dominant negative inhibitor of DARPP-32, Dampinpolypeptides may be expressed in a cell to inhibit PKA activity. In apreferred embodiment of the invention, a Dampin polypeptide orpolynucleotide encoding said polypeptide in used to inhibit PKA activityin a cell. This method comprises the step of: introducing a Dampinpolypeptide or polynucleotide construct comprising an expression controlunit operably linked to a Dampin-encoding polynucleotide into a cell.Preferred cells include but are not limited to: neurons, ovariangranulosa cells, uterine cells, hepatocytes, and myocytes. Methods ofdelivering a polypeptide or polynucleotide construct to a specific celltype are discussed herein. For example, a polynucleotide construct maybe introduced to cells in culture by transfection, electroporation, orviral transduction, as commonly practiced in the art. As a furtherexample, a polynucleotide construct may be introduced to a hepatocyte bypackaging said polynucleotide construct into a liposomal vector;targeting the liposomal vector to the liver by embedding ahepatocyte-specific ligand in the membrane (e.g., hepatocyte growthfactor); and introducing the liposome in a physiologically acceptablemanner to an individual (e.g., orally or by injection). Preferably, thisembodiment is directed toward: decreasing addictive behavior, especiallyin the case of alcohol addiction; reducing cocaine- oramphetamine-dependent responses; reducing progesterone-dependentovulation and egg implantation; or increasing glycogen synthesis tocontrol blood glucose levels, as discussed herein.

In a preferred embodiment of the invention, a substance capable ofdecreasing the ratio of Dampin to DARPP-32 is used in a method toincrease PKA activity in a neuron. Preferred compounds are additionallycapable of passing through the blood brain barrier in vivo. This methodcomprises the step of contacting a neuron with said substance. Deficientdopaminergic signaling (and thus PKA activity) has been implicated inseveral major neurological and psychiatric disorders, includingParkinson's disease, Tourette's syndrome, ADD, Huntington's disease, andschizophrenia. As DARPP-32 is a vital downstream component thedopaminergic pathway, this method is preferably directed towardtreatment of these disorders. Increased activity can be measured by analtered modulation of calcium channel function in response to dopamine,in situ, dopamine-mediated inhibition of the sodium-potassium ATPase(Na,K ATPase) in situ, an increased excitability of striatal andcortical neurons, or dopamine-mediated inhibition of brainsodium-potassium-ATPase activity, as determined in situ. Furthermore,this method may be used to increase sexual receptivity in a femaleindividual. Preferred neurons for use in this method includehypothalamic neurons that express both the dopamine receptor and theprogesterone receptor. Preferred individuals include breeding animals.Further preferred individuals include humans.

In a further preferred embodiment of the invention, a substance thatblocks the inhibition of PKA by Dampin is used in a method to increasePKA activity in a neuron. Preferred compounds are additionally capableof passing through the blood brain barrier in vivo. This methodcomprises the step of contacting a neuron with said substance. Thismethod is directed toward treatment of neurological and psychiatricdisorders, including Parkinson's disease, Tourette's syndrome, ADD,Huntington's disease, and schizophrenia. Furthermore, this method may beused to increase sexual receptivity in a female individual. Preferredneurons for use in this method include hypothalamic neurons that expressboth the dopamine receptor and the progesterone receptor. Preferredindividuals include breeding animals. Further preferred individualsinclude humans.

DARPP-32 is required for proper Atrial Natriuretic Factor (ANF) activityin the kidney. ANF modulates blood sodium levels and reduces bloodvolume by inhibiting the renal sodium-potassium-ATPase, the sole activesodium transporter in the renal basolateral epithelia throughout thenephron. In a preferred embodiment of the invention, a substance capableof decreasing the ratio of Dampin to DARPP-32 expression is used in amethod to activate ANF in a cell. Preferred cells are nephronic kidneycells. This method is applied to inhibit the activity of the renalsodium-potassium-ATPase in response to ANF, as determined in situ, andincrease ANF-mediated sodium excretion in vivo. Preferably, this methodis directed toward decreasing blood volume and hypertension.

In a further preferred embodiment of the invention, a substance thatblocks the inhibition of PKA by Dampin is used in a method to activateANF in a cell. Preferred cells are nephronic kidney cells. This methodis applied to inhibit the activity of the renal sodium-potassium-ATPasein response to ANF, as determined in situ, and increase ANF-mediatedsodium excretion in vivo. Preferably, this method is directed towarddecreasing blood volume and hypertension.

Protein of SEQ SED NO:92 (Internal Designation Clone651658_(—)181-35-2-0-C8-F)

The cDNA of clone 651658_(—)181-35-2-0-C8-F (SEQ ID NO:91) encodes theprotein of SEQ ID NO:92, comprising the amino acid sequence:

MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILESLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQATTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK. Accordingly, itwill be appreciated that all characteristics and uses of polypeptides ofSEQ ID NO:92 described throughout the present application also pertainto the polypeptides encoded by the nucleic acids included in clone651658_(—)181-35-2-0-C8-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:91described throughout the present application also pertain to the nucleicacids included in clone 651658_(—)181-35-2-0-C8-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:91 and SEQ ID NO:92. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO:91 is a novel variant of the human alpha 1anti-trypsin protein named VAGS, encoded by a gene located on chromosome14, specifically at position 14q32.1. The cDNA of SEQ ID NO:91 encodes a418 amino-acid protein of SEQ ID NO:92.

Proteases are key components of a broad range of biological pathways andcan be classified into four groups according to their catalyticmechanisms: the serine, cysteine (thiol), aspartic (carboxyl), andmetalloproteases. VAGS displays serpin motif and thus belongs to theserine protease inhibitor family of protein named serpin. Serpins areirreversible suicide inhibitors of proteases that have a central role inregulating proteolysis in diverse physiological processes such as bloodcoagulation, fibrinolysis, complement activation, angiogenesis,apoptosis, inflammation, neoplasia and viral pathogenesis. VAGSneutralizes any trypsin formed prematurely within the cells by bindingto its active site forming stable complexes with its target enzymes,which is a general property of serpin/serine protease interactions. VAGSis synthesized in the liver and, in response to inflammatory stimuli,inhibits the proteolytic enzyme neutrophil elastase, released fromactivated neutrophils at sites of inflammation. In hepatocytes, VAGSexpression is increased by the cytokine interleukin-6 (IL-6). Synthesisof VAGS is tightly regulated by the net balance of neutrophil elastaseand VAGS at sites of inflammation/tissue injury. Alterations of a serpinwhich can affect its functional levels may result in pathology.Congenital serpin deficiencies cause specific clinical syndromes such asthrombosis with anti-thrombin III deficiency. Individuals with VAGSdeficiency are susceptible to premature development of emphysema andliver diseases. In addition, changes in the balance between serineproteases and their cognate inhibitors may lead to pathological statessimilar to those associated with some neurodegenerative diseases such asAlzheimer's disease.

In one embodiment, VAGS, or fragment thereof, provide an in vitro assayto test the specific sensitivity of various proteases to VAGS. Theprotease inhibitor activity of VAGS may be assessed using any techniquesknown to those skilled in the art including those disclosed in the U.S.Pat. No. 5,955,284, which disclosure is hereby incorporated by referencein its entirety. Possible substrates for the protein of the inventioninclude, but are not limited to, serine proteases such as elastase,trypsin, chymotrypsin, thrombin III, plasmin, heparin, complement II,plasminogen activator, protein C, interleukin-1 beta converting enzyme,preferably trypsin, elastase and chymotrypsin. Methods to assess theactivity of such proteases inhibitors include the steps of contactingthe inhibitor to be tested with one or several protease substrat in acompetition system, and detecting the amount of inhibition of thepresent protein that occurs. Competitive system can also be used todetermine the respective affinities of VAGS among all proteasesubstrates.

In another embodiment, VAGS, or fragment thereof, may be used to remove,identify or inhibit contaminating proteases in a sample. Compositionscomprising the polypeptides of the present invention may be added tobiological samples as a “cocktail” with other protease inhibitors toprevent degradation of protein samples. The advantage of using acocktail of protease inhibitors is that one is able to inhibit a widerange of proteases without knowing the specificity of any of theproteases. Using a cocktail of protease inhibitors also protects aprotein sample from a wide range of future unknown proteases which maycontaminate a protein sample from a vast number of sources. For example,the protein of the invention or fragment thereof are added to sampleswhere proteolytic degradation by contaminating proteases is undesirable.Such protease inhibitor cocktails are widely used in assays to inhibitproteases susceptible of degrading a protein of interest for which theassay is to be performed. Alternatively, the protein of the invention orfragment thereof may be bound to a chromatographic support, either aloneor in combination with other protease inhibitor, using techniques wellknown in the art, to form an affinity chromatography column. A samplecontaining the undesirable protease is run through the column to removethe protease. Alternatively, the same methods may be used to identifynew target proteases of the protein of the invention.

In one embodiment, VAGS, or fragment thereof, may be useful to quantifythe amount of a given protease in a biological sample, and thus used inassays and diagnostic kits for the quantification of proteases in bodilyfluids or other tissue samples, in addition to bacterial, fungal, plant,yeast, viral or mammalian cell cultures. In a preferred embodiment, thesample is assayed using a standard protease substrate. A knownconcentration of protease inhibitor is added, and allowed to bind to aparticular protease present. The protease assay is then rerun, and theloss of activity is correlated to the protease inhibitor activity usingtechniques well known to those skilled in the art. Preferred proteasesin this embodiment are seine protease, more preferably elastase, trypsinand chymotrypsin.

In another preferred embodiment, VAGS, or fragment thereof may be usedas anti-microbial agent useful to inhibit exogenous proteases implicatedin a number of infectious diseases including, but not limited to,bacterial and parasite-borne infections. For example, proteaseinhibitors are able to inhibit growth of all strains of group Astreptococci, including antibiotic-resistant strains. Accordingly, thepresent invention may be used to retard or inhibit the growth of certainmicrobes either in vitro or in vivo.

The present invention provides a method for identify other moleculeswhich specifically binds VAGS. For example, the composition of thebalance proteases/proteases inhibitors of a diseased tissue can bedetermined by isolating the present protein under conditions that do notdisrupt protein-protein interactions, and determining the identity ofproteins associated with the present protein. Such associated proteinscan be identified by any standard method including, but not limited to,immunoprecipitation and immuno-affinity columns. It can also comprise aninvestigation using the yeast-2-hybrid trap for identification of newinteractions involving relevant targets of the present protein thatcould be implicated in some diseases affecting serpin biology. Anothermethod can comprise the combination of the present protein with thelibrary of molecules under conditions suitable to allow complexformation, and detecting complex formation, wherein the presence of thecomplex identifies a molecule which specifically binds the protein ofthe invention and that could be accumulated in some disorders.

In a further embodiment, VAGS provides a method of producing arecombinant serpin capable of effectively modulating serine proteaseactivity. Despite the availability of human alpha anti-trypsin fromserum, quantities large enough for therapeutic uses have beenunobtainable, due in large part to the limited availability of humanserum. Consequently, there is a great need for other sources of alpha 1anti-trypsin to fill the needs created by therapeutic uses. In onepreferred embodiment, milkers animal can be used to produce the proteinof the invention in the milk, thereby generating a significant amount ofthis particular protein after purification. Any type of animal thatproduce enough quantity of milk can be used in this aim such as, but notlimited to, sheep, goat, and cow. These animals can be generated withany method of targeting overexpression of the present protein in themilk. Also in this embodiment, the protein of the invention can beproduced in host cells that have been transfected with an appropriateexpression vector comprising a nucleic acid sequence coding for thepresent protein. The host cells are cultured under conditions wherebythe nucleic acid sequence coding for this particular protein isexpressed. After a suitable amount of time for the product toaccumulate, the protein is purified from the host cells or mediumsurrounding the cells. Introduction of an expression vectorincorporating a nucleic acid sequence coding for the protein of theinvention into a host cell can be performed in a variety of ways, suchas but not limited to calcium or lithium chloride treatment,electroporation, lipofection.

In another embodiment, use of VAGS provides a method of effectivelymodulating serine proteases activity in cells. For example, the level oractivity of the present protein can be increased in cells to decreasethe rate or inhibit specific serine proteases by contacting thebiological sample with an amount of the present protein sufficient todecrease the rate or inhibit specific serine proteases of one or morecells within the sample, or with a compound that increases the activityor expression of the present protein within one or more cells of thesample. Such methods can be performed either in vitro or in vivo. Thelevel of the present protein can be increased in cells in any of anumber of ways, including by administering purified protein to thecells, transfecting the cells with a polynucleotide encoding theprotein, or administering a compound to the cells that causes anincrease in the activity or expression of the protein. Alternatively,serine proteases level can be increase by decreasing the level of thepresent protein in cells, for example using antisense molecules, or morespecifically inhibit the activity of the present protein using direct orindirect inhibitor molecules or antagonistic antibodies directed againstthe present protein.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Such animals are useful for a number ofpurposes, for example to assist with the study of the human alpha 1anti-trypsin deficiency disease, because they represent an in vivo assaymethod for testing candidate molecules potentially useful for thetreatment of various pathophysiological aspects of diseases specificallyrelated to the activity of the present protein. Study of the phenotypeof such models can also allow the identification of additional humanequivalent diseases caused by or linked with alpha 1 anti-trypsindeficiency. These animals can be generated with any method of targetingoverexpression or inactivation of the present protein. Such models areextremely useful, e.g. in the assessment of candidate therapies anddrugs for the treatment of inflammatory diseases and conditions.

In other embodiment, VAGS, or fragment thereof, is used to diagnosediseases or disorders associated with altered expression or activity ofthe present protein. In particular, it is useful in diagnosing patientswith deficient amounts of the present invention which results inuncontrolled activity of target proteases. Examples of such diseases anddisorders include, but are not limited to, alpha 1 anti-trypsindeficiency associated disorders and more specifically liver diseases, ordiseases associated with an excess level of elastase, such as rheumatoidarthritis, emphysema, and psoriasis. The method includes the steps ofcontacting a fluid or tissue sample obtained from an individualsuspected of suffering from the disease or condition, or at risk ofdeveloping the disease or condition, with a compound capable ofselectively binding the present protein or nucleic acids, e.g. apolyclonal or monoclonal antibody or any immunologically active fragmentthereof, a nucleic acid probe, etc., and detecting the level, or anyother detectable property of the present protein in the sample, where adifference in the level or other property in the sample relative to in acontrol sample indicates the presence of the disease or disorder, or ofa propensity for developing the disease or disorder. In this embodiment,the identification of mutations using well known PCR or RT-PCRtechniques and in particular in with real time PCR system that couldfacilitate diagnosis of such conditions. Alternatively, using such amethod, the present invention provides a tool to correlate modulationsin the expression of the specific variant of the invention with somepathologies which have never been linked to. Thus, the present inventionprovides a novel candidate gene for such conditions.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment of diseases andconditions related to the abnormal function of proteases or theirinhibitors. The VAGS, or fragment thereof, may be used to inhibitproteases implicated in a number of diseases where cellular proteolysisoccur such as diseases characterized by tissue degradation preferablyincluding, but not limited to, arthritis, muscular dystrophy,inflammation, tumor invasion, glomerulonephritis, parasite-borneinfections, Alzheimer's disease, periodontal disease, and cancermetastasis. The methods and compositions can also be useful fortreatment of septic shock, pancreatitis, coagulation disorders. In amore preferably embodiment, the invention relates to compositions andmethods to use the protein of the invention or fragment thereof indiseases characterized by an abnormally elevated levels of trypsin,chymotrypsin, or elastase, including but not limited to, chronicemphysema of the lungs, cirrhosis, liver diseases, cystic fibrosis, andmore specifically for alpha 1 anti-trypsin deficiency associateddisorders such as aneurysm or toxic shock. In this embodiment, thepresent invention is preferably applied in the treatment of diseasesassociated with an excess level of elastase, such as rheumatoidarthritis, emphysema, and psoriasis. Indeed, uncontrolled secretion ofelastase which frequently results from aging of the cells or geneticdefects may cause non-specific proteolysis and trigger destructiveprocesses associated with those various chronic diseases. Such methodscomprise the administration of a therapeutically-effective amount of thepresent protein to mammals suffering from the disease or condition,where “effective amount” is meant a concentration of the present proteinwhich is capable of modulating the activity of serine proteases. Thecompositions of the invention are preferably delivered to the affectedmammals in combination with a physiologically acceptable liquid, such asa saline solution or other buffer, or physiologically acceptablecarrier. For treatment of skin inflammation, the compositions of theinvention may be applied to the affected area in combination with aphysiologically acceptable ointment or cream. The proportional ratio ofactive ingredient to pharmaceutical carrier will naturally depend on thechemical nature, solubility, and stability of the recombinant serineprotease inhibitor. The particular amount of the compositions of theinvention that will be administered to the mammal for any particularcondition will depend on the clinical condition of the patient and thetype of illness, and other factors such as the weight, age, the patientand route of delivery. Such composition can be administered by anysuitable route including, but not limited to, intravenous,intramuscular, intraperitoneal, subcutaneous routes, and topically to anaffected area of the skin or by absorption through epithelial ormucocutaneous linings such as nasal, oral, vaginal, rectal.Alternatively, for treatment purposes, the protein of the invention maybe administrated using any of the gene therapy methods known in the art.These compositions can comprise the protein of the invention, and,optionally, one or more other types of protease inhibitors, or any othercompound of interest. Indeed, in this embodiment, the present inventionfind use in drug potentiation applications. For example, therapeuticagents such as antibiotics or antitumor drugs can be inactivated throughproteolysis by endogenous proteases, thus rendering the administrateddrug less effective or inactive. Accordingly, the protease inhibitor ofthe invention may be administrated to a patient in conjunction with atherapeutic agent in order to potentiate or increase the activity of thedrug. This co-administration may be by simultaneous administration, suchas a mixture of the protease inhibitor and the drug, of by separate orsequential administrations. All of these components may be eitherobtained from natural sources or produced by recombinant geneticengineering techniques and/or chemical modification.

Since the regulation of serine proteases by their inhibitors arecritical for the control of tissue destruction in the diseases describedabove, in a further embodiment, VAGS, or fragment thereof provides anassay for the monitoring of markers in vivo for characterisation ofdisease states. The invention thus includes test kits useful for thequantification in a biological sample of the amount of the presentprotein. The kits comprise at least one immunological binding partner,e.g. a monoclonal or polyclonal antibody specific for the protein of theinvention and coupled to detectable markers. In this embodiment, theapplication of such assays can be used to monitor the progress oftherapy administered to treat these or other conditions. Further, theassays can be used as a measure of toxicity, or during clinical testingof new drugs to assess the impact on tissue degradation. Thus the assaysmay be applied in any situation wherein the present invention can beused as an index of the condition, treatment, or effect of substancesdirectly administered to the subject or to which the subject is exposedin the environment. This marker may thus also play a role as prognosticindicators, preferably concerning inflammatory diseases. For example, itcan be used in the Alzheimer's disease where chronic inflammation is anaccompanying physiological contributor to this multifactor pathology.Also in a preferred embodiment, the present invention provides a methodof detecting the presence and/or monitoring the metastatic progress of amalignancy. Indeed, metastatic potential can be influenced by theinteraction between the neoplastic cells and their microenvironment suchas extracellular matrix and proteolytic enzymes including the presentprotein. The invention thus includes test kits useful for quantify theamount of the present protein in a biological sample comprising thesteps of contacting the biological sample with a specific monoclonal orpolyclonal antibody specific for the present protein and coupled todetectable markers. Thus, the condition of a patient can be monitoredcontinuously and the quantified amount of such proteins measured in thepathological sample can be compared with the amount quantified in abiological sample of a normal individual or with the previous analysisof the same patient. In all this embodiment, this marker can be measuredeffectively in plasma, serum or blood, by any suitable method, includingimmunoassays. It can also preferably be mesured in tissues and fluidsrecovered from inflammatory sites. Thus, the condition of a subject canbe monitored continuously and the quantified amount of this particularprotein measured in the pathological sample can be compared with theamount quantified in a biological sample of a normal individual.

Polynucleotides of SEQ ID NO:93 (Internal Designation Clone150011_(—)110-006-3-0-D5-F) and SEQ ID NO:95 (Internal Designation Clone500737461_(—)205-43-3-0-E3-F)

The cDNA of clone 150011_(—)110-006-3-0-D5-F (SEQ ID:93) encodes anallele of Tissue Factor Pathway Inhibitor-1 (TFPI-1), comprising thenucleotide sequence:

CTCTTTGCTCTAACAGACAGCAGCGACTTTAGGCTGGATAATAGTCAAATTCTTACCTCGCTCTTTCACTGCTAGTAAGATCAGATTGCGTTTCTTTCAGTTACTCTTCAATCGCCAGTTTCTTGATCTGCTTCTAAAAGAARAAGTAGAGAAGATAAATCCTGTCTTCAATACCTGGAAGGAAAAACAAAATAACCTCAACTCCGTTTTGAAAAAAACATTCCAAGAACTTTCATCAGAGATTTTACTTAGATGATTTACACAATGAAGAAAGTACATGCACTTTGGGCTTCTGTCCCTGCTGCTTAATCTTGCCCCTGCCCCTCTTAATGCTGATTCTGAGGAAGATGAAGAACACACAATTATCACAGATACGGAGTTGCCACCACTGAAACTTATGCATTCATTTTGTGCATTCAAGGCGGATGATAGCCCATGTAAAGCAATCATGAAAAGATTTCTTCAATATTTTCACTCGACAGTGCGAAGAATTTATATATGGGGGATGTGAAGGAAATCAGAATCGATTTGAAAGTCTGGAAGAGTGCAAAAAAATGTGTACAAGAGATAMTGCAAACAGGATTATAAAGACAACATTGCAACAAGAAAAGCCAGATTTCTGCTTTTTGGAAGAAGATCCTGGAATATGTCGAGGTTATATTACCAGGTATTTTTATAACAATCAGACAAAACATGTGAACGTTTCAAGTATGGTGGATGCCTGGGCAATATGAACAATTTTGAGACACTGGAAGAATGCAAGAACATTTGTGAAGATGGTCCGAATGGTTTCCAGGTGGATAATTATGGAACCCAGCTCAATGCTGTGAATAACTCCCTGACTCCGCAATCAACCAAGGTTCCCAGCCTTTTTGTTACAAAAGAAGGAACAAATGATGGTTGGAAGAATGCGGCTCATATTTACCAAGTCTTTYTGAACGCCTTCTGCATTCATGCATCCATGTTCTTTCTAGGATTGGATAGCATTTCATGCCTATGTTAATATTTGTGCTTTTGGCATTTCCTTAATATTTATATGTATACGTGATGCCTTTGATAGCATACTGCTAATAAAGTTTTAATATTTACATGCATAGGAAAAA AAAAAAAAAA.Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:94 and polynucleotides of SEQ ID NO:93described throughout the present application also pertain to the nucleicacids included in Clone 150011_(—)110-006-3-0-D5-F. Clone150011_(—)110-006-3-0-D5-F is alternatively referred to herein asTFPI-C16Pfs in reference to the nucleotide polymorphism that is asubject of the present invention. A preferred embodiment of theinvention is directed toward the compositions of SEQ ID NO:93, SEQ IDNO:94, and Clone 150011_(—)110-006-3-0-D5-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

The cDNA of clone 500737461_(—)205-43-3-0-E3-F (SEQ ID:95) encodes anallele of Tissue Factor Pathway Inhibitor-1 (TFPI-1), comprising thenucleotide sequence:

CTCTTTGCTCTAACAGACAGCAGCGACTTTAGGCTGGATAATAGTCAAATTCTTACCTCGCTCTTTCACTGCTAGTAAGATCAGATTGCGTTTCTTTCAGTTACTCTTCAATCGCCAGTTTCTTGATCTGCTTCTAAAAGAAGAAGTAGAGAAGATAAATCCTGTCTTCAATACCTGGAAGGAAAAACAGAATAACCTCAACTCCGTTTTGAAAAAACATTCCAAGAACTTTCATCAGAGATTTTACTTAGATGATTTACACAATGAAGAAAGTACATGCACTTTGGGCTTCTGTATGCCTGCTGCTTAATCTTGCCCCTGCCCCTCTTAATGCTGATTCTGAGGAAGATGAAGAACACACAATTATCACAGATACGGAGTTGCCACCACTGAAACTTATGCATTCATTTTGTGCATTCAAGGCGGATGATGGCCCATGTAAAGCAATCATGAAAAGATTTTTCTTCAATATTTTCACTCGACAGTGCGAAGAATTTATATATGGGGGATGTGAAGGAAATCAGAATCGATTTGAAAGTCTGGAAGAGTGCAAAAAATGTGTACAAGAGATAATGCAAACAGGATTATAAAGACAACATTGCAACAAGAAAAGCCAGATTTCTGCTTTTTGGAAGAAGATCCTGGAATATGTCGAGGTTATATTACCAGGTATTTTTATAACAATCAGACAAAACAGTGTGAACGTTTCAAGTATGGTGGATGCCTGGGCAATCAACAATTTTGAGACACTGGAACAATGCAAGAACATTTGTGAAGATGGTCCGAATGGTTTCCAGGTGGATAATTATGGAACCCAGCTCAATGCTGTGAATAACTCCCTGACTCCGCAATCAACCAAGGTTCCCAGCCTTTTTGAATTTCACGGTCCCTCATGGTGTCTCACTCCAGCAGACAGAGGATTGTGTCGTGCCAATGAGAACAGATTCTACTACAATTCAGTCATTGGGAAATGCCGCCCATTTAAGTACAGTGGATGTGGGGGAAATGAAAACAATTTTACTTCCAAACAAGAATGTCTGAGGGCATGTAAAAAAGGTTTCATCCAAAGAATATCAAAAGGAGGCCTAATTAAAACCAAAAGAAAAAGAAAGAAGCAGAGAGTGAAAATAGCATATGAAGAAATTTTTGTTAAAAATATGTGAATTTGTTATAGCAATGTAACATTAATTCTACTAAATATTTTATATGAAATGTTTCACTATGATTTTCTATTTTTCTTCTAAAATGCTTTTAATTAATATGTTCATTAAATTTTCTATGCTTATTGCAAAAAAAAAAAAAAAA. Accordingly, it will be appreciatedthat all characteristics and uses of the polynucleotides of SEQ ID NO:95and polypeptides of SEQ ID NO:96 described throughout the presentapplication also pertain to the nucleic acids included in Clone500737461_(—)205-43-3-0-E3-F. Clone 500737461_(—)205-43-3-0-E3-F isalternatively referred to herein as TFPI-M162Qfs in reference to thenucleotide polymorphism that is a subject of the present invention. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:95, SEQ ID NO:96, and Clone500737461_(—)205-43-3-0-E3-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The extrinsic coagulation pathway is initiated on exposure of Tissuefactor (TF) to plasma (McVey J H, Bailliere's Clinical Haematology12:361-72 (1999) which disclosure is hereby incorporated by reference inits entirety). Tissue Factor Pathway Inhibitor-1 (TFPI-1) is a negativeregulator of the extrinsic coagulation pathway (U.S. Pat. No. 5,849,875which disclosure is hereby incorporated by reference in its entirety).

TFPI-1 is a secreted trivalent Kunitz-type plasma proteinase inhibitor.TFPI-1 negatively regulates the initiation of coagulation through amechanism of activated factor X (FXa) feedback inhibition of thecatalytic complex of activated factor VII (FVIIa) and TF. That is,TFPI-1 directly inhibits FXa and, in a FXa-dependent fashion, producesfeedback inhibition of the TF-FVIIa complex by allosteric enablement ofTF-FVIIa binding. TFPI-1 is the major inhibitor of the protease activityof the TF-FVIIa complex. The second Kunitz domain of TFPI-1 binds andinhibits FXa, whereas the first Kunitz domain is responsible for theinhibition of FVIIa in the TF-FVIIa complex. The function of the thirdKunitz domain is unknown, although there is evidence that it contains aheparin binding site. Heparin binding site(s) have also been mappedcarboxyl-terminal to the third Kunitz domain.

Tissue factor pathway of coagulation plays a dominant role during normalhaemostasis. TFPI-1, expressed primarily by the microvascularendothelium, appears to be the major physiologic inhibitor of TF-inducedcoagulation. TF-initiated coagulation also plays an important role inthe pathophysiology of many diseases, including coronary thrombosis,disseminated intravascular coagulation, stroke, and atheriosclerosis.Several animal studies have found a beneficial effect of recombinantTFPI-1 in some of these clinical conditions.

TFPI-1 plays an important role in modulating TF-dependentthrombogenesis. Recombinant full-length TFPI-1 prevents thrombosisformation and rethrombosis after lysis in a rabbit model of jugular veinthrombosis (Kaiser, B et al. Thromb. Haemost. 76:615-20 (1996) whichdisclosure is hereby incorporated by reference in its entirety). In arat model of disseminated intravascular coagulation, TFPI-1 was found toinhibit thrombus formation (Elsayed, Y A et al., Am. J. Clin. Pathol.106:574-83 (1996) which disclosure is hereby incorporated by referencein its entirety).

High levels of TF antigen and activity are detected in atherioscleroticlesions, particularly in the advanced lesions. When the plaques areruptured or eroded, exposure of cellular and extracellular TF tocirculating blood plays a pivotal role in mediating fibrin-rich thrombusformation leading to acute coronary syndromes. Presence of TFPI-1 inatheriosclerotic plaques is associated with reduced tissue factoractivity and reduced plaque thrombogenicity (Caplice, N M et al.,Circulation 98:1051-7 (1998); Badimon, J J et al., Circulation 99:1780-7(1999) which disclosures are hereby incorporated by reference in theirentirety).

An recent study in mice using the gene knockout technology unambiguouslyestablished that deficiency of TFPI-1 promotes atheriosclerosis andthrombosis. In this work, it was found that TFPI-1 protects fromatheriosclerosis and is an important regulator of the thrombosis thatoccurs in the setting of atheriosclerosis (Westrick, R J et al.,Circulation, 103:3044-6 (2001) which disclosure is hereby incorporatedby reference in its entirety). Importantly, it was found that in thismodel inactivation of only one of the two copies of the TFPI-1 gene wassufficient to promote atheriosclerosis and thrombosis.

Recently several amino acid polymorphisms have been identified for humanTFPI-1. A mutation at nucleotide position 1 of exon 7 results in thesubstituion of leucine for proline at position 179 (numbered from theinitiating methionine of TFPI-1) (Kleesiek, K et al., Blood 10:3976-7(1998) which disclosure is hereby incorporated by reference in itsentirety). This mutation occurs immediately downstream of Kunitz domain2 (U.S. Pat. No. 5,849,875 which disclosure is hereby incorporated byreference in its entirety). This mutation has been found to bestatistically associated with a higher risk for venous thrombosis(Kleesiek, K et al., Thromb. Haemost. 82:1-5 (1999) which disclosure ishereby incorporated by reference in its entirety).

A second amino acid polymorphism results in the substitution ofmethionine for valine at position 292 (numbered from the initiatingmethionine of TFPI-1). This mutation occurs very near thecarboxy-terminus of TFPI-1. As might be expected for a mutation so fardownstream of Kunitz domains 1 and 2, no link was found between thismutation and venous thromboembolic disease (Arnaud, E et al., Thromb.Haemost. 82:159-60 (1999) which disclosure is hereby incorporated byreference in its entirety).

The cDNA of clone 150011 encodes the protein of SEQ ID NO:94. In thecase of TFPI-C16Pfs, a deletion of two nucleotides in codon 16 (numberedfrom the initiating methionine of TFPI-1) results in the substitution ofproline for cysteine and in the introduction of a frame-shift leading topremature termination of the protein within the signal sequence (exon3). Specifically, whereas codon 16 of TFPI-1 reads TGC (U.S. Pat. No.5,849,875 which disclosure is hereby incorporated by reference in itsentirety), in TFPI-C16Pfs nucleotides T and G have been deleted. Asprotein TFPI-C16Pfs terminates upstream of Kunitz domains 1 and 2, theprotein of SEQ ID NO:94 is nonfunctional.

The cDNA of clone 500737461 encodes the protein of SEQ ID NO:96. In thecase of TFPI-M162Qfs, a deletion of two nucleotides in codon 162(numbered from the initiating methionine of TFPI-1) and mutation of theremaining nucleotide results in the substitution of glutamine formethiomine and in the introduction of a frame-shift leading to prematuretermination of the protein within Kunitz domain 2 (exon 6).Specifically, whereas codon 162 of TFPI-1 reads ATG (U.S. Pat. No.5,849,875 which disclosure is hereby incorporated by reference in itsentirety), in TFPI-M162Qfs two of the nucleotides have been deleted andthe third changed to C. As protein TFPI-Ml62Qfs terminates within Kunitzdomain 2, neither FXa binding nor the consequential enablement ofTF-FVIIa-binding by Kunitz domain 1 occurs, leading to nonfunctionalprotein of SEQ ID NO:96.

The availability of informative genetic screenings and diagnosticmarkers for genetic predisposition to thrombosis would be ofconsiderable value. On one hand, said information can be used by thepatient to make appropriate lifestyle changes. On the other hand, saidinformation can be used by the physician to anticipate thromboticcomplications that might arise in the course of clinical procedures. Inboth cases, said information results in health benefit to the patientand in reduced medical costs borne by the patient as well as by societyin general.

The nucleotide polymorphisms that are described herein for clones 150011(TFPI-C16Pfs) and 500737461 (TFPI-M162Qfs) and that are the subject ofthe present invention lead to nonfunctional TFPI-1. There is clearevidence that having even just one of the two copies of the TFPI-1 geneinactivated predisposes the patient to atheriosclerosis and thrombosis.It follows therefore that the nucleotide polymorphisms described herewithin the coding region of TFPI-1 that lead to nonfunctional TFPI-1have genetic screening and diagnostic value in identifying patients thatare genetically predisposed to atheriosclerosis and thrombosis.

In a preferred embodiment, the present invention provides for a methodof diagnosing genetic predisposition to atheriosclerosis and thrombosisthrough the identification of a dinucleotide deletion in TFPI-1 codon 16(numbered from the initiating methionine of TFPI-1). Methods ofidentifying such a dinucleotide deletion are well known to those skilledin the art and include, but are not restricted to, to PCR-SSCP(polymerase chain reaction followed by single-strand conformationpolymorphism) (Kleesiek, K et al., Blood 10:3976-7 (1998) whichdisclosure is hereby incorporated by reference in its entirety).

In a further preferred embodiment, the present invention is drawn to amethod of determining if an individual is at increased risk ofdeveloping atheriosclerosis and thrombosis comprising the step ofidentifying a dinucleotide deletion in TFPI-1 codon 16 (numbered fromthe initiating methionine of TFPI-1), preferably using the method ofPCR-SSCP, in a biological sample, preferably blood, wherein saiddeletion indicates increased risk.

In additional preferred embodiment, the present invention provides for amethod of diagnosing genetic predisposition to atheriosclerosis andthrombosis through the identification of a dinucleotide deletion inTFPI-1 codon 162 (numbered from the initiating methionine of TFPI-1).Methods of identifying such a dinucleotide deletion are well known tothose skilled in the art and include, but are not restricted to, toPCR-SSCP (polymerase chain reaction followed by single-strandconformation polymorphism) (Kleesiek, K et al., Blood 10:3976-7 (1998)which disclosure is hereby incorporated by reference in its entirety).

In a further preferred embodiment, the present invention is drawn to amethod of determining if an individual is at increased risk ofdeveloping atheriosclerosis and thrombosis comprising the step ofidentifying a dinucleotide deletion in TFPI-1 codon 162 (numbered fromthe initiating methionine of TFPI-1), preferably using the method ofPCR-SSCP, in a biological sample, preferably blood, wherein saiddeletion indicates increased risk.

Protein of SEQ ID NO:100 (Internal Designation Clone479155_(—)174-4-4-0-C8-F)

The cDNA of clone 479155_(—)17-4-4-4-0-C8-F (SEQ ID NO:99) encodes theprotein of SEQ ID NO:100 comprising the amino acid sequence

MIVKGVASRTVVSRPFPGNWLFSSIQLTDDQGPVLMTTVAMPVFSKQNETRSKGILLGVVGTDVPVKELLKTIPKYKLGIHGYAFAITNNGYILTHPELRLLYEEGKKRRKPNYSSVDLSEVEWEDRDDVLRNAMVNRKTGKFSMEVKKTVDKGVHFSQTFLLLNLKQTTVKN. Accordingly itwill be appreciated that all characteristics and uses of polypeptides ofSEQ ID NO:100 described throughout the present application also pertainto the polypeptides encoded by the nucleic acids included in Clone479155_(—)174-4-4-0-C8-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:99described throughout the present application also pertain to the nucleicacids included in Clone 479155_(—)174-4-4-0-C8-F. A preferred embodimentof the invention is directed toward the compositions of SEQ ID NO:99,SEQ ID NO:100, and Clone 479155_(—)174-4-4-0-C8-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments. The protein of SEQ IDNO:100 encodes ADEVAR, a variant of calcium channel alpha2delta3 subunitresulting from alternative splicing. ADEVAR has novel function asdescribed below.

Alpha2delta3 subunit is a component of voltage-gated Ca2+ channels. Thealpha2 subunit has several hydrophobic sequences, but biosyntheticstudies indicate that it is an extracellular, extrinsic membrane proteinattached to the membrane through disulfide linkage to the delta subunit.The delta subunit is encoded by the 3′end of the coding sequence of thesame gene as the alpha2 subunit, and the mature forms of these twosubunits are produced by post-translational proteolytic processing anddisulfide linkage (Catterall, W A, Annu. Rev. Cell Dev. Biol. 16:521-55(2000) which disclosure is hereby incorporated by reference in itsentirety). Alpha2delta3 subunit is expressed exclusively in the brain(Klugbauer, N et al., J. Neuroscience 19:684-691 (1999) which disclosureis hereby incorporated by reference in its entirety). Alpha2delta3subunit plays a role in regulating neuronal Ca2+ currents (Catteral, WA, Annu. Rev. Cell Dev. Biol., 16:521-55 (2000); Stefani, A et al.,Neuropharmacology 37:83-91 (1998) which disclosures are herebyincorporated by reference in their entirety). Alpha2delata3 subunit hasbeen implicated in epileptic seizures (Gee NS et al., J. Biol Chem.271:5768-76 (1996); Bryans JS et al., J. Med. Chem. 41:1838-1845 (1998)which disclosures are hereby incorporated by reference in theirentirely).

ADEVAR is a product of alternative splicing leading to a soluble proteintruncated at both it amino- and carboxyl-termini. ADEVAR plays anegative regulatory role in Ca2+ channel function. Diminished ADEVARexpression leads to dysregulated Ca2+ flux through the channel andreduced neuronal excitability.

In a preferred embodiment, the present invention provides for anantibody that specifically binds ADEVAR of the present invention.Further preferred is a method of making said antibody wherein saidantibody recognizes a non-conformational or conformational epitope ofADEVAR.

Further preferred is a method wherein a mouse is immunized with ADEVAR.Further preferred is said immunization with ADEVAR, wherein ADEVAR isproduced by recombinant DNA methodology. Further preferred is a methodwherein monoclonal antibodies from said mouse are screened for bindingto ADEVAR but not to full-length alpha2delta3 subunit. Further preferredis a method wherein monoclonal antibodies derived from said mouse arescreened by enzyme-linked immunosorbent assay (ELISA) for binding toADEVAR but not to full-length alpha2delta3 subunit. Methods ofexpressing protein by recombinant DNA methodology are well known tothose skilled in the art. Methods of generating said monoclonal antibodyand of establishing specificity by methods including ELISA are wellknown to those skilled in the art.

In a further preferred embodiment, the present invention provides for amethod wherein said ADEVAR antibody is used in a method of quantitatingADEVAR in bodily fluid. Further preferred is a method of quantitatingADEVAR in bodily fluid, wherein the method of quantitation is a sandwichELISA format. Further preferred is a method wherein said ADEVAR antibodyis used to measure ADEVAR concentration in cerebrospinal fluid. In apreferred embodiment, the present invention provides for a method ofcontacting said antibody and specifically binding it with ADEVAR.Further preferred is a method for using said antibody diagnostically tostratify seizures and thereby add value to therapeutic strategies.Further preferred is a method of diagnosis, wherein reduced ADEVAR levelis associated with predisposition to seizure in a subset of patientsmanifesting seizure.

Protein of SEQ ID NO:102 (Internal Designation Clone586587_(—)181-9-2-0-C5-F)

The cDNA of Clone 586587_(—)181-9-2-0-C5-F (SEQ ID NO:101) encodes hABCof SEQ ID NO:102, comprising the amino acid sequence:

MACWPQLRLLLWKNLTFRRRQTCQLLLEVAWPLFIFLILISVRLSYPPYEQHECHFPNKAMPSAGTLPWVQGIICNANNPCFRYPTPGEAPGVVGNFNKSIVARLFSDARRLLLYSQKDTSMKDMRKVLRTLQQIKKSSSRGDKRHFLNWQKGLKPLPQALL. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:102 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone586587_(—)181-9-2-0-C5-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NOs:101described throughout the present application also pertain to the nucleicacids included in Clone 586587_(—)181-9-2-0-C5-F. A preferred embodimentof the invention is directed toward the compositions of SEQ ID NO:102,SEQ ID NO:101, and Clone 586587_(—)181-9-2-0-C5-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments. hABC is a novel splicevariant of the ATP-binding cassette 1. As a splice variant, hABC is only162 amino-acid long whereas ABCA1 is 2261 amino acid long. hABC displays100% identity with ABCA1 over its 140 amino-terminal residues, whereasthe 22 carboxyl-terminal amino acids are unique to hABC. hABC does notdisplay the Walker A and B motifs nor the active transport signature.The 140 common amino acids correspond to the cytoplasmic amino-terminaltail of ABCA1 that plays a role in cholesterol-binding. Furthermore,hABC displays one transmembrane domain (TCQLLLEVAWPLFIFLILISV) and a“positive-hydrophobic-polar” signal peptide that is required fortranslocation to the plasma membrane. Moreover, the hABC splice variantis specifically expressed in liver cells. Thus, hABC plays an importantrole in clearing HDL from the bloodstream by binding to HDL-cholesterol,thus allowing HDL-cholesterol import to liver cells where lipids arecatabolized and excreted.

An embodiment of the invention is directed to a composition comprising ahABC polypeptide sequence of SEQ ID NO:102.

A further embodiment of the invention is directed to a compositioncomprising a hABC polypeptide fragment having biological activity.

An embodiment of the invention is directed to a composition comprising apolynucleotide sequence of SEQ ID NO:101 encoding a hABC polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a hABC polypeptidefragment having biological activity.

An embodiment of the invention is directed to a composition comprising apolynucleotide sequence that yields an RNA that is complementary to thesequence of SEQ ID NO:101 encoding a hABC polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence that yields an RNA that iscomplementary to a polynucleotide sequence encoding a hABC polypeptidefragment. Preferred such a polynucleotide sequence is the polynucleotidesequence that yields an RNA that is complementary to

GAGGGGACAAACGCCATTTCCTCAACTGGCAGAAGGGACTGAAGCCTCTCCCTCAAG CCCTTTTA.

A further embodiment of the invention is directed to compositionscomprising an antibody directed against a hABC polypeptide or against ahABC polypeptide fragment having the same biological activity.Preferably, the antibody specifically binds to the hABC polypeptide orand not to the ABCA1 polypeptide. Even more preferably, the antibodyrecognizes the LQQIKKSSSRGDKRHFL amino-acid sequence or theRHFLNWQKGLKPLP amino-acid sequence.

An embodiment of the present invention relates to methods of measuringthe circulating HDL-cholesterol in bodily fluids. Methods of detectingmeasuring the circulating HDL-cholesterol comprise the steps of i)labeling by standard methods of the hABC polypeptide with a moleculewhich can be used to provide a quantifiable signal, ii) addition of thisprobe, under conditions suitable for the formation of hybridizationcomplexes, to a fluid obtained from a patient and to control fluidscontaining a known amount of HDL-cholesterol, iii) washing of thesamples, after a suitable incubation period, in order to remove all hABCpolypeptides that are not complexed with HDL-cholesterol, and iv)comparison of the resulting signal with control samples containing aknown amount of HDL-cholesterol. Such methods can be used in diagnostickits for detecting diseases associated with low circulatingHDL-cholesterol level, for evaluating the efficacy of a particulartherapeutic treatment regimen in animal studies and in clinical trials,and for monitoring the treatment of an individual patient. The bindingefficiency of hABC to HDL-cholesterol can be determined using anytechnique familiar to those skilled in the art, e.g. using the assaydescribed in U.S. Pat. No. 5,962,322, which disclosure is herebyincorporated by reference in its entirety.

An embodiment of the present invention relates to compositionscomprising an antibody directed against hABC or fragment thereof, and toa method to decrease uptake of HDL-cholesterol comprising the step ofinhibiting hABC binding to HDL-cholesterol using an anti-hABC antibody.Preferably, such compositions comprise the preferred antibodiesdescribed above. Such compositions can be administered to a cell, atissue sample or a patient. Preferably, this method is directed totreating an individual with low circulating HDL-cholesterol level bydecreasing HDL-cholesterol clearance.

Another embodiment relates a method to decrease uptake ofHDL-cholesterol comprising the step of inhibiting hABC expressionwithout affecting ABCA1 expression using an antisense polynucleotide. Insuch a method, recombinant expression vectors comprising a polypeptidethat yields an RNA that is complementary to the sequence of the hABCmRNA can be administered to cell, a tissue sample or a patient.Preferred such an antisense polynucleotide is described above. Preferredexpression vectors include viral vectors, especially adenoviral andlentiviral vectors. Preferably, the antisense polynucleotides of thepresent invention are administered to hepatocytes.

In another embodiment, genetic modification of a cell with a vectorcomprising a polynucleotide that yields an RNA that is complementary tothe sequence of the hABC mRNA may be accomplished using one or moretechniques well known in the gene therapy field. For example, one of themethods described in Mulligan (Mulligan, Science, 260:926-32 (1993)),which disclosure is hereby incorporated by reference in its entirety,can be used. Preferably, such a method is directed to treating anindividual with low circulating HDL-cholesterol level by decreasingHDL-cholesterol clearance.

A further embodiment of the present invention is directed to substancesthat decrease hABC expression without affecting ABCA1 expression, and toa method of screening for such substances comprising the steps of: i)contacting a cell with a test substance, ii) comparing hABC expressionin the cell after exposure to that of an unexposed control cell, iii)comparing ABCA1 expression in the cell after exposure to that of anunexposed control cell, iv) quantifying said expression levels, and v)determining the ratios of hABC and ABCA1 expression in an exposed cellrelative to the expression in an unexposed cell. Preferably, hABCexpression is studied in an hepatocyte and ABCA1 expression is studiedin a macrophage.

In another preferred embodiment, compositions comprising substances thatdecrease hABC expression without affecting ABCA1 expression can beadministered to patients presenting low levels of HDL-cholesterol.

Additionally, the compositions comprising an antibody directed againsthABC or substances that decrease hABC expression without affecting ABCA1expression can be processed in accordance with conventional methods ofpharmacy to produce medicinal agents for administration to patients. Forexample, the pharmaceutical composition comprising an antibody directedagainst hABC substances that decrease hABC expression without affectingABCA1 expression may be made up in a solid form (e.g. granules for oraladministration, powders for inhalation) or in a liquid form (e.g.solutions for oral administration or for injection).

Effectiveness of the compositions can be verified in vivo by measuringthe plasma HDL-cholesterol level of an animal model before and afteradministration of the composition of the present invention. Thecirculating HDL-cholesterol level can for example be measured using thefast pressure liquid chromatography technique as described in U.S. Pat.No. 5,962,322, which disclosure is hereby incorporated by reference inits entirety. The dosage regimen for treating a human patient presentinglow circulating HDL-cholesterol with compositions of the presentinvention may vary widely, but can be determined using standard methods.For example, the amount of antibody directed against or substances thatdecrease hABC expression without affecting ABCA1 expression is an amountsufficient to increase circulating low HDL-cholesterol in the plasma ofa subject.

The compositions of the invention may be administered alone or incombination with other known agents increasing circulatingHDL-cholesterol level, e.g., gemfibrozil, niacin and the SR-BIHDL-cholesterol receptor. Diseases associated with low circulatingHDL-cholesterol level that may be treated by compositions and methods ofthe present invention include, but are not limited to, artherosclerosis,angioplasty, dyslipidemia associated with non insulin-dependant diabetesmellitus, obesity and various other coronary artery diseases.

Protein of SEQ ID NO:104 (Internal Designation Clone620315_(—)188-13-1-0-G12-F)

The cDNA of Clone 620315_(—)188-13-1-0-G12-F (SEQ ID NO:103) encodesMOBP-81h of SEQ ID NO:104, comprising the amino acid sequence:

MSQKPAKEGPRLSKNQKYSEHFSIHCCPPFTFLNSKKEIVDRKYSICKSGCFYQKKEEDWICCACQKTRLKRKIRPTPKKK. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:104 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone620315_(—)188-13-1-0-G12-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NOs:103described throughout the present application also pertain to the nucleicacids included in Clone 620315_(—)188-13-1-0-G12-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:104, SEQ ID NO:103, and Clone 620315_(—)188-13-1-0-G12-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of the present invention, named MOBP-81h, is a novel splicevariant of the myelin-associated oligodendrocytic basic protein (MOBP,Genbank accession number BAA05659). MOBP-81h is only 81 amino acidslong, whereas MOBP is 183 amino acids long. The first exon is identicalbetween the two cDNAs. MOBP-81h lacks the second exon of MOBP, and thetwelve carboxyl-terminal amino acids of MOBP-81h are unique to thissplice variant. MOBP-81h, is the first splice variant described for thehuman MOBP protein. MOBP-81h is specifically expressed in CNSoligodendrocytes, and plays a role in maintaining myelin sheathintegrity.

An embodiment of the invention is directed to a composition comprising aMOBP-81h polypeptide sequence of SEQ ID NO:104.

A further embodiment of the invention is directed to a compositioncomprising a MOBP-81h polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:103 encoding aMOBP-81h polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a MOBP-81h polypeptidefragment having biological activity.

In another embodiment, the compositions of the present inventioncomprise MOBP-81h polypeptides. The method of producing MOBP-81hpolypeptides comprises the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any techniquewell-known to those skilled in the art. Preferably, an antibody directedagainst MOBP-81h or fragment thereof may be bound to a chromatographicsupport to form an affinity chromatography column. Even more preferably,the antibody recognizes the twelve carboxyl-terminal amino acids ofMOBP-81h.

An embodiment of the present invention relates to methods of using thepolypeptides and the polynucleotides of the present invention to treator to reduce in severity demyelinating disorders. Any compositions andmethods containing, e.g., MOBP-81h polypeptide or fragment thereof, apolynucleotide encoding the protein, or a compound that increases theexpression or activity of MOBP-81h can be used.

In an embodiment, the methods of the present invention relate to theadministration of a recombinant expression vector comprising one of thepolynucleotides of the invention to a patient suffering from ademyelinating disease. Preferred expression vectors include viralvectors, especially adenoviral and lentiviral vectors.

In another embodiment, genetic modification of a cell with a vectorcomprising one of the polynucleotides of the invention may beaccomplished using one or more techniques well known in the gene therapyfield. For example, one of the methods described in Mulligan (Mulligan,Science, 260:926-32 (1993)), which disclosure is hereby incorporated byreference in its entirety, can be used.

In still another embodiment, the compositions of the present inventioncomprise a substance that increases MOBP-81h expression.

Additionally, the methods of the present invention relate to methods ofscreening test substances that increase MOBP-81h expression. Thesemethods comprise the steps of: i) contacting a cell with a testsubstance; and ii) comparing MOBP-81h expression in the cell afterexposure to the test substance to that of an unexposed control cell.Preferably, the test substance modifies the expression of MOBP-81h inoligodendrocytes while not in other cell types.

Effectiveness of compositions and methods of the present invention totreat demyelinating diseases can be verified in vitro by studying theeffects of the compositions of the present invention on the morphologyof myelin sheaths by immunoelectron microscopy. Effectiveness ofcompositions and methods of the present invention to treat demyelinatingdiseases can be verified in vivo using experimental models ofdemyelinating disorders, e.g., TMEV-infected mice. Effective doses ofthe polypeptides or polynucleotides of the present invention fortreating a patient suffering from demyelinating disorders can bedetermined according to the relevant techniques. For example, theeffective amounts of compositions of the present invention can bedetermined by measuring the necessary and sufficient amount ofcomposition for disappearance or reduction in severity of clinicalmanifestations associated with demyelinating disorders (e.g. tremor,tonic seizure, unstable locomotion, ataxia).

In a preferred embodiment, MOBP-81h polypeptides or a substance thatincreases MOBP-81h expression can be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients. Thus, the pharmaceutical compositioncomprising MOBP-81h or fragment thereof or a substance that increasesMOBP-81h expression may be made up in a solid form (e.g. granules fororal administration, powders for inhalation) or in a liquid form (e.g.solutions for oral administration or for injection).

The compositions of the invention may be administered alone or incombination with other known agents treating demyelinating disorders,e.g., imidazol derivatives or MBP molecules. Demyelinating disordersthat may be treated by a composition containing MOBP-81h or fragmentthereof include but are not limited to leukodystrophies (e.g. Krabbe'sdisease, metachromatic leukodistrophy, ALD, Canavan disease, Alexanderdisease), leukoencephalopathies, multiple sclerosis and virus-inducedinflammatory demyelination.

Protein of SEQ ID NO:106 (Internal Designation Clone646477_(—)181-19-2-0-F4-F)

The cDNA of Clone 646477_(—)181-19-2-0-F4-F (SEQ ID NO:105) encodesnovel Apolipoprotein H (NAPOH) of SEQ ID NO:106, comprising the aminoacid sequence:

MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWLINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMFGNDTITCTTHGNWTKLPECREVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTKLGNWSAMPSCKASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWKTDASDVKPC. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:106 described throughout the present application also pertain tothe polypeptides encoded by the nucleic acids included in Clone646477_(—)181-19-2-0-F4-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:105described throughout the present application also pertain to the nucleicacids included in Clone 646477_(—)181-19-2-0-F4-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:105, SEQ ID NO:106, and Clone 646477_(—)181-19-2-0-F4-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:106 is a polymorphic variant of the sequence ofapolipoprotein H or beta-2-glycoprotein I precursor (swissprot accessionnumberP02749). Like apoliprotein H, the protein of the inventiondisplays 4 Sushi domains (PF00084) and one sushi-like domain, from aminoacids 23 to 79 (Sushi 1), amino acids 84 to 137 (Sushi 2), amino acids142 to 200 (Sushi 3), amino acids 205 to 260 (Sushi 4) and amino acids263 to 345 (Sushi-like). Sushi domains are also known as Complementcontrol protein (CCP) modules, or short consensus repeats (SCR), existin a wide variety of complement and adhesion proteins. Also, it has beenreported that the domain V (sushi-like domain) specifically interactswith hydrophobic ligands (Hong, D. P. et al., Biochemistry 40:8092-8100(2001)). Novel apolipoprotein H, the protein of SEQ ID NO:106, is highlyexpressed in liver.

Novel apolipoprotein H is a plasma protein with the ability to bind withvarious kinds of negatively charged substances. Novel apolipoprotein H(NAPOH) may prevent activation of the intrinsic blood coagulationcascade by binding to phospholipids on the surface of damaged cells.NAPOH is a strong auto-antigen that stimulates a vigorous B cell-humoralresponse and T cell immunity response. NAPOH has been implicated in avariety of physiologic pathways including lipoprotein metabolism,artherosclerosis and in the production of antiphospholipidautoantibodies (“aPA”). NAPOH also binds to platelets, mitochondria,heparin, DNA, and anionic phospholipids, and has been shown to beinvolved in the blood coagulation pathway, platelet aggregation, andprothrombinase acitvity of platelets. NAPOH exerts multiple inhibitoryeffects on the coagulation pathway and platelet aggregation. NAPOH isconsidered to be a required cofactor for anionic phospholipids antigenby the aPA found in sera of many patients with chronic inflammatorydisease, like systemic lupus erythematosus, and primary antiphospholipidsyndrome, but it does not seem to be required for the reactivity of aPAassociated with infections. These studies suggest that theNAPOH-phospholipid compex forms the antigen to which aPA are directed.Autoantibodies to phospholipid-free NAPOH are present in patients withprimary antiphospolipid syndrome. Antiphospholipid autoantibodies are aheterogeneous group of autoantibodies including most commonly a lupusanticoagulant and anticardiolipin antibodies which are directed againstnegatively charged phospholipids. The presence of antiphospholipidautoantibodies has been associated with recurrent deep vein thrombosisand other thrombotic complications, including pulmonary, renal, andretinal thrombosis, as well as Budd-Chiari syndrome. In addition,antiphospholipid autoantibodies have been associated with arterialthrombosis including cerebral, retinal, and peripheral arteries.Recurrent fetal losses, usually occurring in the second and thirdtrimester, felt to be due in part to thrombosis of the placental vesselsand subsequent infarction resulting in placental insufficiency andultimately fetal loss are associated with antiphospholipidautoantibodies.

An embodiment of the invention is directed to a composition comprising anovel Apolipoprotein H (NAPOH) polypeptide sequence of SEQ ID NO:106.

A further embodiment of the invention is directed to a compositioncomprising a NAPOH polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:105 encoding a NAPOHpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NAPOH polypeptidefragment having biological activity.

Preparation and purification of the protein of SEQ ID NO:106 orfragments thereof may be carried out as described in U.S. Pat. No.5,859,213, the disclosure of which is incorporated herein by referencein its entirety. For example, a method of purifying NAPOH from humanblood plasma comprising the steps: i) heating and cooling the plasma toobtain a precipitate and a supernatant, ii) separating the supernatantand acidifying the supernatant, iii) adding a precipitation agent to thesupernatant and separating aqueous albumin solution from secondprecipitate, iv) subjecting the aqueous albumin solution to affinitychromatography; and v) eluting the particulate support to obtain NAPOH.

A further embodiment of the invention is directed to a method ofscreening test substances for activators or inhibitors of NAPOHexpression comprising the steps of: i) contacting a cell with a testsubstance; and ii) comparing NAPOH expression in the cell after exposureto the test substance to that of an unexposed control cell. As a result,such NAPOH activators are of great potential as new drugs due to theirability to induce coagulation and are expected to be useful in treatmentof various coagulation disorders (including but not limited tohereditary disorders, such as hemophilias and disseminated intravascularcoagulation, a severe hemorragic syndrome) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. Alternatively, such NAPOH inhibitors can beuseful in treatment of autoimmune diseases and thrombotic diseases.

A further embodiment of the invention is directed to a method ofscreening for test substances that specifically bind to NAPOH andprevent binding to antiphospholipid autoantibodies to comprising thesteps of: i) contacting a test substance with NAPOH polypeptide in thepresence of antiphospholipid autoantibodies, under conditions that allowbinding of NAPOH to antiphospholipid autoantibodies and ii) detectingthe amount of antiphospholipid autoantibodies bound to NAPOH in thepresence and absence of the test substance by methods common to the art.Preferably, the test substance is able to inhibit NAPOH interaction withantiphospholipid autoantibodies. Interaction of NAPOH withautoantibodies is linked to antiphopoholipid syndrome and morespecifically to autoimmune artherogenesis.

A further embodiment of the invention is directed to a method ofscreening substances for modulators of NAPOH expression comprising thesteps of: i) contacting a cell with a test substance; and ii) comparingNAPOH expression in the cell after exposure to the test substance tothat of an unexposed control cell. NAPOH expression is determined bymethods common to the art or included herein, by detecting NAPOHpolynucleotides or polypeptides. An example of this method comprises thestep of: i) culturing two equivalent cell samples; ii) adding a testsubstances to one of the cultures and not the other; iii) harvestingboth cultures at a specified time; iv) purifying the mRNA from eachsample of cells; v) comparing the level of NAPOH mRNA in each sample byNorthern Blot, RTPCR, or another method common to the art. The inventionprovides for design and use of specific polynucleotide probes andprimers, as described herein. An additional example comprises the stepof: i) having two equivalent cultures of cells; ii) adding a testsubstance to one of the cultures and not the other; iii) harvesting bothcultures; iv) purifying the protein from each sample of cells; v)comparing the level of NAPOH polypeptides in each sample by Westernblot, immunohistochemistry, or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein. As a result, such NAPOHactivators are of great potential as new drugs due to their ability toinduce coagulation and are expected to be useful in treatment of variouscoagulation disorders (including but not limited to hereditarydisorders, such as hemophilias and disseminated intravascularcoagulation, a severe hemorragic syndrome) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. Alternatively, such NAPOH inhibitors can beuseful in treatment of autoimmune diseases and thrombotic diseases bydecreasing the level of inflammatory aPAs. In another embodiment, theinvention relates to methods for using the protein of the invention orfragments to identify autoantibodies which are related to autoimmunedisease and systemic lupus erythematosus (SLE). Accordingly, the presentprotein may be used to detect the presence of autoantibodies. In atypical embodiment, the protein of SEQ ID NO:106 is labeled with anydetectable moiety including, but are not limited to, a fluorescentlabel, a radioactive atom, a paramagnetic ion, biotin, achemiluminescent label or a label which can be detected through asecondary enzymatic or binding step. The invention further provides amethod of diagnosing SLE, and distinguishing such processes from otherdiseases.

An antagonist of the protein of SEQ ID NO:106 may be produced usingmethods which are generally know in the art. The antagonist will affectthe binding activity of NAPOH to negatively charged phopholipids whichare implicated in autoimmune disorders. In one aspect, the protein ofthe invention or a fragment thereof may be used to synthesize specificantibodies using any techniques known to those skilled in the artincluding those described therein. In particular, purified NAPOH may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind NAPOH. NAPOH may thusbe used for the characterization and assay of antibody against thisprotein in patients suffering from autoimmune disorder.

The ability of the protein of the invention or fragment thereof tofunction as a major antigen for antiphospholipid antibodies may beassessed using techniques well known to those skilled in the art. Theability of the protein of the invention or fragment thereof, especiallyfragments containing Sushi motifs and Sushi-like motifs, to bind toantiphospholipid autoantibodies may be assessed using techniques wellknown to those skilled in the art including those described herein. Forexample, the protein of SEQ ID NO:106 or a fragment thereof may be fixedto a solid support, such as a chromatograpy matrix. A preparationcontaining antiphopholipid autoantibodies is placed in contact with theprotein of the invention under conditions which facilitate binding toNAPOH. The support is washed and then the antiphopholipid autoantibodiesare released from the support by contacting the support with agentswhich cause antiphopholipid autoantibodies to dissociate from the NAPOH.

An embodiment of the present invention relates to methods of using theprotein of the invention or fragment thereof particularly polypeptidescontaining Sushi motifs, or derivative thereof to identify and/orquantify binding autoantibodies, preferably anti phospholipidautoantibodies, in a biological sample, and thus used in assays anddiagnostic kits for the quantification of such binding proteins inbodily fluids, in tissue samples, and in mammalian cell cultures. Suchassays may be particularly useful as diagnostic or prognostic tools inthe detection and monitoring of a disorder linked to primaryantiphospholipid syndrome. The binding activity of the protein of theinvention or fragment thereof may be assessed using any method familiarto those skilled in the art. Preferably, a defined quantity of theprotein of the invention or fragment thereof is added to the sampleunder conditions allowing the formation of a complex between the proteinof the invention or fragment thereof and the binding protein to beidentified and/or quantified. Then, the presence of the complex and/oror the free protein of the invention or fragment thereof is assayed andeventually compared to a control using any of the techniques known bythose skilled in the art.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of SEQ ID NO:105 or fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations.The microarray can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents (see for example: Chee, M. et al., Science,274:610-614 (1996) which disclosure is hereby incorporated by referencein its entirety). For example, it has been shown that genetic variants,mutations, and polymorphisms are related to thrombotic related diseaseand chronic inflammatory disease (described in U.S. Pat. Nos. 6,203,980B1, the disclosure of which is incorporated herein by reference in itsentirety).

In addition, NAPOH is involved in the fertilization process. Theaddition of the purified protein to prepared sperm samples fromnormospermic men increases significantly the straight line velocity(VSL) and the amplitude of lateral head displacement (ALH). Storage ofsperm is of widespread importance in commercial animal breedingprograms, human sperm donor programs, and in the treatment of certaindisease states. For example, sperm samples may be frozen for men whohave been diagnosed with cancer or other diseases that may eventuallyinterfere with sperm production, as well as for assisted reproductionpurposes where sperm may be stored for use at other locations or times.The procedures utilized in such cases include: washing a sperm sample toseparate out the sperm-rich fraction from non-sperm components of asample such as seminal plasma or debris; further isolating the healthy,motile sperm from dead sperm or from white blood cells in an ejaculate;freezing or refrigerating of sperm for use at a later date or forshipping to females at differing locations; extending or diluting spermfor culture in diagnostic testing or for use in therapeuticinterventions such as in vitro fertilization or intracytoplasmic sperminjection (Cohen et al. 12: 994-1001 (1997)). Once sperm have beenwashed or isolated, they are then extended (or diluted) in culture orholding media for a variety of uses (sperm analysis, diagnostic tests,assisted reproduction). Each of these uses for extended or diluted spermrequires a somewhat different formulation of basal medium (see, forreview, U.S. Pat. No. 6,140,121 Ellington et al. October 2000); however,in all cases sperm survival is suboptimal outside of the femalereproductive tract. Novel additional components of a dilution or storagemedium which could improve the functional preservation of sperm would beuseful. Therefore, in another preferred embodiment of this invention,purified recombinant proteins encoded by SEQ ID NO:106 or fragmentsthereof can be added as components of pharmacological media designed toprotect spermatozoa. The methods used to compose such preservation mediaare generally known by those skilled in the art (for example, Oliver S.A., et al. U.S. Pat. No. 5,897,987 April 1999; Cohen J. et al., supra).Inversely, in yet another embodiment of this invention, ligands,inhibitors, neutralizing antibodies or other biological agents whichrecognize the protein of the invention and which bind it and which blockit can be used as components of pharmacological formulations designedfor male contraception purposes.

Protein of SEQ ID NO:108 (Internal Designation Clone113165_(—)105-056-3-0-G12-F)

The cDNA of clone 113165 (SEQ ID NO:107) encodes the protein of SEQ IDNO:108, comprising the amino acid sequence:

MAAGGSGVGGKRSSKSDADSGFLGLRPTSVDPALRRRRRGPRNKKRGWRRLAQEPLGLEVDQFLEDVRLQERTSGGLLSEAPNEKLFFVDTGSKEKGLTKKRTKVQKKSLLLKKPLRVDLILENTSKVPAPKDVLAHQVPNAKKLRRKEQLWEKLAKQGELPREVRRAQARLLNPSATRAKPGPQDTVERPFYDLWASDNPLDRPLVGQDEFFLEQTKKKGVKRPARLHTKPSQAPAVEVAPAGASYNPSFEDHQTLLSAAHEVELQRQKEAEKLERQLALPATEQAATQESTFQELCEGLLEESDGEGEPGQGEGPEAGDAEVCPTPARLATTEKKTEQQRRREKAVHRLRVQQAALRAARLRHQELFRLRGIKAQVALRLAELARRQRRRQARREAEADKPRRLGRLKYQAPDIDVQLSSELTDSLRTLKPEGNILRDRFKSFQRRNMIEPRERAKFKRKYKVKLVEKRAF REIQL.Accordingly, it will be appreciated that all characteristics and uses ofpolypeptides of SEQ ID NO:108 described throughout the presentapplication also pertain to the polypeptides encoded by the nucleicacids included in clone 113165_(—)105-056-3-0-G12-F. In addition, itwill be appreciated that all characteristics and uses of thepolynucleotides of SEQ ID NO:107 described throughout the presentapplication also pertain to the nucleic acids included in clone113165_(—)105-056-3-0-G12-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:107, SEQ ID NO:108, andclone 113165_(—)105-056-3-0-G12-F. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding the fragments.

The cDNA of SEQ ID NO:107 is a novel human JNK3-binding protein namedhJNK3-BP, homologous to a murine JNK3-binding protein (GSP:AAB12882).The cDNA of SEQ ID NO:107 encodes a 478 amino-acid protein of SEQ IDNO:108, which is predominantly expressed in the brain.

The c-Jun NH2-terminal kinase (JNK) signal transduction pathway isactivated in response to various environmental stress and by theengagement of several classes of cell surface receptors. In mammaliancells, JNK has been implicated in the immune response, oncogenictransformation and apoptosis. These effects of JNK are mediated, atleast in part, by increased gene expression. Three mammalian genesencode JNK protein kinases. JNK1 and JNK2 are expressed ubiquitously,while JNK3 is expressed primarily in the brain (Ip YT, Davis R J CurrOpin Cell Biol 1998 April;10(2):205-19). By performing a yeasttwo-hybrid screen specifically with JNK3 as a bait, Ito M, et al. (ItoM, Yoshioka K, Akechi M, Yamashita S, Takamatsu N, Sugiyama K, Hibi M,Nakabeppu Y, Shiba T, Yamamoto K I. Mol Cell Biol 1999November;19(11):7539-48) isolated mouse Jsap1 (for JNK/stress-activatedprotein kinase-associated protein 1), also known as Jip-3 (Kelkar N,Gupta S, Dickens M, Davis R J, Mol Cell Bio 2000 20:1030-1043). Jip-3represents a JNK-interacting proteins (JIPs), such as Jip-1 and Jip-2,acting as scaffolding proteins that may regulate signal transduction bythe JNK signaling pathway. The protein of the invention hJNK3-BPspecifically binds JNK3 protein kinase, modulating the biologicaleffects of JNK3 signaling pathway in cells. hJNK3-BP represents thefounding member of a new class of scaffold protein involved in theregulation of the JNK3 cascade.

An embodiment of the invention is directed to a composition comprising ahJNK3-BP polypeptide sequence of SEQ ID NO:108.

A further embodiment of the invention is directed to a compositioncomprising a hJNK3-BP polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:107 encoding ahJNK3-BP polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a hJNK3-BP polypeptidefragment having biological activity.

In one embodiment, the present invention provides a method of producinga recombinant protein capable of effectively modulating JNK activity.The protein of the invention can be produced in host cells that havebeen transfected with an appropriate expression vector comprising anucleic acid sequence coding for hJNK3-BP polypeptides. Introduction ofan expression vector incorporating a nucleic acid sequence coding forthe protein of the invention into a host cell can be performed in avariety of ways, including but not limited to calcium or lithiumchloride treatment, electroporation, or lipofection. Any of a widevariety of expression systems can be used to provide the recombinantproteins. Suitable expression vehicles include, but are not limited toplasmids, viral particles and baculovirus for insect cells. Theexpression vehicle can be integrated into the host cell genome. In somecircumstances, it is desirable to employ an inducible expression vector.The host cells harboring the expression vehicle are cultured inconventional nutrient media, under conditions whereby the nucleic acidsequence coding for this particular protein is expressed. After asuitable amount of time for the product to accumulate, the protein ispurified from the host cells.

In another embodiment, the present invention provides a method ofeffectively modulating JNK activity in cells. The level or activity ofhJNK3-BP can be increased in cells to decrease or inhibit specific JNKprotein kinase activity, thereby preventing JNK3-associated apoptosis.hJNK3-BP levels may be increased by introducing hJNK3-BP polynucleotidesor polypeptides into a cell in an amount sufficient to specificallyinhibit JNK protein kinase activity of one or more cells within thesample. Such methods can be performed either in vitro or in vivo. Thelevel of hJNK3-BP can be increased in cells in any of a number of ways.For instance, purified hJNK3-BP protein may be introduced to the cellsby microinjection or by liposome or micelle-mediated transport. Suchliposomal or micellar microcapsule may optionally be combined with acell type-specific target, such as an antibody or receptor ligand.Alternatively, hJNK3-BP polynucleotides may be introduced to a cell bymethods common to the art such as transfection, electroporation, orviral transduction. Cyclodextrin, liposome or micelle-mediated transportmay also be used to introduce hJNK3-BP polynucleotides to a cell. Usefulexamples of the above methods are described in U.S. Pat. Nos. 5,019,369,5,616,565, 6,110,490, 6,204,060, and P.C.T. WO9704748, disclosures ofwhich are hereby incorporated by reference in their entireties. Inaddition, any compound that increases the expression of hJNK3-BPpolypeptides can be used to decrease JNK protein kinase activity withinone or more cells of the sample. Such compounds can be identified byscreening for test substances that increase hJNK3-BP expressioncomprising the steps of: contacting a cell with a test substance andcomparing hJNK3-BP expression in the cell after exposure to the testsubstance to that of an unexposed control cell.

The present invention provides an in vitro method to inhibit apoptosisinduced by JNK activation to keep cells alive in culture. Preferably,the present invention is suited to the culturing of cells for purposesincluding transplantation or implantation of such cells in vivo after anex vivo introduction of hJNK3-BP polynucleotides. Said polynucleotidesmay be introduced to a cell of interest by methods known in the art,such as those listed above. Furthermore, such a method can be used withneurons or other cell types which undergo apoptosis in culture.Transplantation of healthy neurons expressing a hJNK3-BP into subjectswhose neurons are degenerating can alleviate some effects of theneuronal diseases or disorders. Treated cells can be grafted, inparticular, into the brain, either as cells cultured in vitro on asupport matrix using techniques disclosed in the U.S. Pat. No.6,264,943, which disclosure is hereby incorporated by reference in itsentirety, or as dispersed cells.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Preferably, the expression of hJNK3-BPpolypeptides is targeted in the brain. The transgene can be integratedas a single transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene can also be selectively introducedinto and activated in a particular cell type using a conditionalexpression system. Such animals are useful for a number of purposes,because they represent an in vivo assay method for testing candidatemolecules potentially useful for the treatment of variouspathophysiological aspects of diseases specifically related to theactivity or consequence of the activity of hJNK3-BP polypeptides on JNKbiological effects. Study of the phenotype of such models can also allowthe identification of additional human diseases associated with JNKabnormal activity. These animals can be generated with any method oftargeting overexpression or inactivation of hJNK3-BP to produce thefounder lines of transgenic animals. Such models are extremely useful,e.g. in the assessment of candidate therapies and drugs for thetreatment of neurodegenerative diseases and autoimmune or malignancyconditions.

In other embodiment, the protein of the invention or fragment thereof isused to diagnose diseases or disorders associated with abnormal hJNK3-BPactivity and in particular with altered JNK biological effects. Inparticular, it is useful in diagnosing patients with deficient amountsof hJNK3-BP which results in uncontrolled activity of JNK protein kinaseand monitor hJNK3-BP expression in such conditions. Preferably, thepresent invention provides a method of diagnose pathologies linked toaltered apoptosis or inflammatory responses such as, but are not limitedto, neurodegenerative diseases characterized by apoptosis, includingParkinson's disease and Alzheimer's disease, autoimmune diseases such asarthritis or other conditions characterized by inflammation andmalignancies such as leukemias. The method comprises the steps ofcontacting a tissue sample obtained from an individual suspected ofsuffering from the disease or condition or at risk of developing thedisease or condition, with a detectably labeled compound capable ofselectively binding hJNK3-BP polypeptides or nucleic acids. For example,a polyclonal or monoclonal antibody or any immunologically activefragment thereof or a nucleic acid probe may be used.

This marker may thus also play a role as prognostic indicators,preferably concerning inflammatory diseases. More preferably, it can bemeasured in tissues and fluids recovered from inflammatory sites. Thus,the condition of a subject can be monitored continuously and thequantified amount of this particular protein measured in thepathological sample can be compared with the amount quantified in abiological sample of a normal individual or with previous samples of thesame patient.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment or prevention ofdiseases and conditions related to the abnormal JNK biological effectsand preferably with abnormal apoptosis. The protein of the invention orfragment thereof may be used to treat neurodegenerative diseasescharacterized by apoptosis, including Parkinson's disease andAlzheimer's disease. Other conditions that can be treated using thecompositions and methods of the invention are autoimmune diseases suchas arthritis, other conditions characterized by inflammation such asinflammatory arthritis and bronchial asthma, and malignancies such as,but not limited to leukemias.

In another embodiment of the present invention is to provide novelmethods and compositions useful for the treatment or prevention ofdiseases and conditions associated with oxidative damage dependent onabnormal JNK biological effects. The protein of the invention orfragment thereof can be used to treat or prevent oxidative damage toorgans such as the liver and kidney, and in particular, damage due toischemia/reperfusion in heart disease and cardiomyopathy. Morepreferably, such methods and compositions can also be used to treatdonor organs for transplantation. Indeed these organs are exposed tosubstantial environmental stress which can affect the normal functioningof the organs; effects of which can be blocked by JNK modulators such ashJNK3-BP.

Such methods comprise the administration of a therapeutically-effectiveamount of hJNK3-BP polypeptides to mammals suffering from the disease orcondition, where “effective amount” is meant a concentration of hJNK3-BPpolypeptides which is capable of modulating JNK biological effects. Thecompositions of the invention are preferably delivered to an individualin combination with a pharmaceutically acceptable carrier, such as asaline solution or other physiological buffer suitable foradministration to a patient. The particular amount of the compositionsof the invention that will be administered to the mammal for anyparticular condition will depend on the clinical condition of thepatient, and other factors such as the weight, age, and route ofdelivery. Such composition can be administered by any suitable route.Alternatively, for treatment purposes, nucleic acids can be administeredto the patient using any of the standard vectors and/or gene deliverymethods known in the art. Suitable gene delivery systems include, butare not limited to liposomes, naked DNA and viral vectors. Thesecompositions can comprise the protein of the invention, and, optionally,one or more other compounds of interest. Indeed, in this embodiment, thepresent invention find use in drug potentiation applications. Thisco-administration may be by. simultaneous administration or by separateor sequential administrations. All of these components may be eitherobtained from natural sources or produced by recombinant geneticengineering techniques and/or chemical modification.

Protein of SEQ ID NO:110 (Internal Designation Clone231462_(—)117-065-1-0-G11-F)

The cDNA of Clone 231462_(—)117-065-1-0-G11-F (SEQ ID NO:109) encodesthe 386 amino acid long polypeptide, DROCK2, of SEQ ID NO:110 comprisingthe amino acid sequence:

MCLLLSCPCHPSAHGQSMWIERTSFVTAYKLPGILRWFEVVHMSQTTISPLENAIETMSTANEKILMMINQYQSDETLPINPLSMLLNGIVDPAVMGGFAKYEKAFFTEEYVRDHPEDQDKLTHLKDLIAWQIPFLGAGIKIHEKRVSDNLRPFHDRMEECFKNLKMKVEKEYGVREMPDFDDRRVGRPRSMLRSYRQMSIISLASMNSDCSTPSKPTSESFDLELASPKTPRVEQEEPISPGSTLPEVKLRRSKKRTKRSSVVFADEKAAAESDLKRLSRKHEFMSDTNLSEHAAIPLKASVLSQMSFASQSMPTIPALALSVAGIPGLDEANTSPRLSQTFLQLSDGDKKTLTRKKVNQFFKTMLASKSAEEGKQIPDSLSTDL. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:110 describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in Clone231462_(—)117-065-1-0-G11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:109described throughout the present application also pertain to the nucleicacids included in Clone 231462 117-065-1-0-G11-F. A preferred embodimentof the invention is directed toward the compositions of SEQ ID NO:109,SEQ ID NO:110, and Clone 231462 117-065-1-0-G11-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

DROCK2, the protein of SEQ ID NO:110, is a splicing variant of DOCK2(EMBL entry Q92608) retaining the last 370 amino acid of DOCK2, whilethe first sixteen amino acids (MCLLLSCPCHPSAHGQ) represent specificDROCK2 amino acids corresponding to signal sequence. The resultingisoform is thus lacking the N-terminal sequences of the DOCK2 isoform.However, it retains DOCK2's C-terminal domain comprising a twenty aminoacid sequence (LASKSAEEGKQIPDSLSTDL) which has been shown to be involvedin protein-protein interactions by interacting with PDZ domain ofmembrane-associated proteins.

DROCK2 belongs with DOCK2 to the CDM family of signaling proteins whichalso comprises the human DOCK180 protein and its homologues, the Ced-5protein in Caenorhabditis elegans and Mbc polypeptide in Drosophilamelanagaster. These proteins share extensive similarities at the aminoacid level, except in their carboxyl-terminal regions that aredivergent. CDM proteins have been implicated in polarized extension ofthe cell surface in their respective organisms. Mbc in Drosophila isnecessary for myoblast fusion and for migration of epithelial cells,both of which require reorganization of the cytoskeleton. Ced-5 has alsobeen shown to be involved in the regulation of the cytoskeleton in thenematode, loss of function of which results in defects in engulfing deadcells and in the migration of distal tip cells. Finally, the humanDOCK180 protein which was originally identified as one of the majorproteins bound to the CrkII adaptator protein, is involved in membraneruffling and cell migration in nonadherent cells. It has been shown totransduce signals from the CrII-p130Cas complex to both the cytoskeletonand JNK pathway by activating the low molecular weight Rac GTPase.

DROCK2, in contrast to DOCK180, which is expressed in all tissues exceptin peripheral blood cells, is expressed only in circulating blood cells,lymphocytes and macrophages present in organs. Thus the protein isspecifically expressed by nonadherent cells. DROCK2 is involved in bloodcell migration and phagocytosis of apoptotic cells by macrophages whereit binds to and activates Rac GTPAses.

An embodiment of the invention is directed to a composition comprising aDROCK2 polypeptide sequence of SEQ ID NO:110.

A further embodiment of the invention is directed to a compositioncomprising a DROCK2 polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:109 encoding a DROCK2polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a DROCK2 polypeptidefragment having biological activity.

A further embodiment of the invention is directed to a method ofscreening test substances for modulators of DROCK2 expression comprisingthe steps of: i) contacting a cell with a substance to be tested; andii) comparing DROCK2 expression in the cell after exposure to the testsubstance to that of an untreated control cell.

In one embodiment, the present invention provides a method of producinga recombinant protein capable of effectively increasing Rac GTPaseactivity. The protein of the invention can be produced in host cellsthat have been transfected with an appropriate expression vectorcomprising a nucleic acid sequence coding for the protein of theinvention. Introduction into a host cell of such expression vector forDROCK2 can be performed in a variety of ways, including but not limitedto calcium or lithium chloride treatment, electroporation, orlipofection. Any of a wide variety of expression systems can be used toprovide the recombinant proteins. Suitable expression vehicles include,but are not limited to plasmids, viral particles or baculovirus forinsect cells. The expression vehicle can be integrated into the hostcell genome. Optionally, an inducible expression vector can be used toachieve tight controlled expression of the gene in the host cell.

Another embodiment the present invention provides methods to purify fromcellular extracts proteins harboring one or more PDZ domain, preferablyproteins belonging to the MAGUK (Membrane Associated and GuanylateKinase) family, more preferably proteins selected from the groupconsisting of DLG, syntenin or PSD95 proteins, by using the presentprotein, preferably its C-terminal twenty amino acid sequence tocopurify those proteins. Methods to affinity purify proteins are wellknown for those skilled in the art. For example, the PDZ-containingproteins can be purified on an affinity column or on solid support likebeads using the polypeptides of the invention. The protein to bepurified using the present method can be derived from any source, e.g.protein expressed in vitro using an invertebrate, yeast or bacterialheterologous expression system.

Another embodiment of the invention is directed to a method to increasephagocytosis of apoptotic cells. Preferably, this method is applied invivo to an individual. The method comprises the steps of: i) removing asample of monocytes, ii) introducing a polynucleotide encoding a DROCK-2polypeptide or fragment thereof ex vivo to those cells, and iii)reinjecting the recombinant cells into an individual. Using such amethod in combination with anticancer or antiviral therapies would be ofparticular interest for the rapid elimination of apoptotic cancer orinfected cells.

An embodiment of the invention provides for a method of screening testsubstances for modulators of DROCK-2 expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing DROCK-2 expression in the cell after exposure to the testsubstance to that of an unexposed control cell. DROCK-2 expression isdetermined by methods common to the art or included herein, by detectingDROCK-2 polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of DROCK-2 mRNA ineach sample by Northern blot, RTPCR, or another method common to theart. The invention provides for design and use of specificpolynucleotide probes and primers, as discussed herein. An additionalexample comprises the steps of: i) having two equivalent cultures ofcells; ii) adding a test substance to one of the cultures and not theother; iii) harvesting both cultures; iv) purifying the protein fromeach sample of cells; v) comparing the level of DROCK-2 polypeptides ineach sample by Western blot, immunohistochemistry, or another methodcommon to the art. The invention provides for design and use of specificantibodies and antibody fragments, as discussed herein. Substances thatincrease DROCK-2 expression (agonists) may be used to increasecytoskeletal remodeling and Rac activation. Substances that decreaseDROCK-2 expression (antagonists) may be used to inhibit cytoskeletalremodeling and Rac activation. Methods utilizing DROCK-2 agonists andantagonists are included herein.

A preferred embodiment of the invention provides a method of screeningfor test substances that bind DROCK-2 polypeptides. This methodcomprises the steps of: i) contacting a test substance with a DROCK-2polypeptide or fragment thereof under conditions that allow binding; andii) detecting the binding of the test substance by methods common to theart (e.g., competitive antibody-based methods such ascommunopreciptation and Western blotting). Included in this method aretest substances that are conjugated to an antibody, antibody fragment,cell-type specific ligand or a portion thereof.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to DROCK-2 for antagonists ofDROCK-2 activity. This method comprises the steps of: i) contacting acell with a test substance; and ii) comparing DROCK-2 biologicalactivity after exposure to the test substance to that of an unexposedcontrol cell. Detection of DROCK-2 biological activity may be detectedby detecting activity of Rac GTPase. An example of an assay detectingRac activity comprises the steps of: exposing Rac GTPase to radiolabeledGTP and detecting the amount of hydrolysis by detecting the amount offree, radiolabeled phosphate.

A further embodiment of the present invention is also directed to amethod to reduce the elimination rate of apoptotic cells in a patientsubjected to an antiapoptotic treatment, such method comprising removinga sample of the monocytes/macrophages of said patient, inhibiting orreducing the expression of the present protein in the isolated cells exvivo, and reinjecting the modified cells to the patient. Methods toinhibit the expression of a given gene in a cell are well known in theart, e.g. using antisense or ribozyme strategies, any of which can beused in the present method. Alternatively, reduced phagocytosis ofapoptotic cells in said patient can be achieved by interfering with thenormal activity of the present protein. In such a method, the isolatedmonocytes are transfected ex vivo with a DROCK2 fragment correspondingto the last twenty carboxy-terminal amino acid prior to reinjection intothe patient. Because reducing phagocytosis of apoptotic cellsconcomitantly with the administration of the antiapoptotic agent wouldhelp maintaining more dying cells alive and therefore available for theaction of the antiapoptotic agent, such method would be of particularinterest to increase the treatment efficiency of diseases associatedwith abnormal cell apoptosis, including but not limited toneurodegenerative disorders.

A preferred embodiment provides a method of preventing and treatinginvasive neoplasms that require cytoskeletal remodeling (e.g., forextravasation). This method comprises the step of contacting anantagonist of DROCK-2 expression or activity with a cell. Preferredcells include nonadherent cells. Further preferred cells includelymphocytes and macrophages. Preferably, the DROCK-2 antagonist isdelivered to a specific cell type, for example, by conjugating theantagonist to a cell-type specific targeting moiety (e.g., a ligand orantibody fragment). DROCK-2 antagonists in a physiologically acceptablesolution may be delivered by methods common to the art, such as orallyor parenterally. This method is useful for prevention and treatment ofleukemias and other invasive neoplasms.

Protein of SEQ ID NO:112 (Internal Designation Clone500723589_(—)205-34-3-0-G4-F)

The cDNA of clone 500723589_(—)205-34-3-0-G4-F (SEQ ID NO:111) encodesNovel 17 beta-hydroxysteroid dehydrogenase type 2 (NBHSD2) of SEQ IDNO:112, comprising the amino acid sequence:

MSTFFSDTAWICLAVPTVLCGTVFCKYKKSSGQLWSWMVCLAGLCAVCLLILSPFWGLILFSVSCFLMYTYLSGQELLPVDQKAVLVTGGDCGLGHALCKYLDELGFTVFAGVLNENGPGAEELRRTCSPRLSVLQMDITKPVQIKDAYSKVAAMLQDRGLWAVINNAGVLGFPTDGELLLMTDYKQCMAVNFFGTVEVTKTFLPLLRKSKGRLVNVSSMGGGAPVERLASYGSSKAAVTMFSSVMRLELSKWGIKVASIQPGGFLTNIAGTSDKWEKLEKDILDHLPAEVQEDYCQDYILAQRNFLLLINSLASKDFSPVLRDIQHAILAKSPFAYYTPGKGAYLWICLAHYLPIGIYDYFAKRHFGQDKPMPRALRMPNYKKKAP. Accordingly, it will be appreciated thatall characteristics and uses of the polypeptides of SEQ ID NO:112described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in Clone500723589_(—)205-34-3-0-G4-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:111described throughout the present application also pertain to the nucleicacids included in Clone 500723589_(—)205-34-3-0-G4-F. A preferredembodiment of the invention is directed toward the compositions of SEQID NO:111, SEQ ID NO:112, and clone 500723589_(—)205-34-3-0-G4-F. Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of SEQ ID NO:112 is a polymorphic variant of the sequence of17 beta estradiol dehydrogenase (swissprot accession number P37059).Like 17 beta-hydroxysteroid dehydrogenase type 2, the protein of theinvention displays a short chain dehydrogenase domain PF00106) spanningfrom positions 83 to 268, a ferredoxin domain (PS00197) spanning frompositions 40 to 48 and an ADH-short domain spanning from positions 219to 247.

Novel 17 beta-hydroxysteroid dehydrogenase type 2 (NBHSD2) is an enzymeof the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family.The 17 beta-hydroxysteroid dehydrogenases are pivotal in controlling thebiological potency of steroid hormones by catalyzing oxidation orreduction at position 17.

17 Beta-hydroxysteroid dehydrogenases catalyze the interconversionbetween high-activity 17beta-hydroxysteroids and low-activity17-ketosteroids. Because both estrogens and androgens have the highestaffinity towards their receptors in the 17 beta-hydroxy form, the 17beta-HSD enzymes regulate the biological activity of sex hormones.Several 17beta-HSD may metabolize further substrates including alcohols,bile acids, fatty acids and retinol. The activities of 17 beta HSDs areessential for gonadal sex steroid biosynthesis and they are alsoinvolved in the modulation of steroid hormone action in peripheraltissues. This family of steroidogenic enzymes constitutes an interestingtarget in the control of the concentration of estrogens and androgenssince this family is involved in the formation and inactivation of sexsteroids.

NBHSD2 catalyzes the oxidative reaction or the inactivation of sexsteroids thereby reducing the exposure of tissues to the action of sexsteroids. NBHSD2 preferentially catalyzes the oxidation of estradiol(E(2)) to inactive estrogen, estrone (E(1)), testosterone to 4-dione,dihydrotestosterone (DHT), 20alpha-dihydroprogesterone (20alpha-DHP),and androst-5-ene-3, 17-diol (5-diol) to DHEA with NAD+ as the coenzyme.Therefore, NBHSD2 is involved in the regulation of clearance and/ormetabolism of sex steroids.

Local formation of sex steroids plays a major role in both normal andneoplastic hormone-sensitive tissues. 40% of all cancers, namely,breast, prostate, ovarian and uterine cancers, are sex steroid-sensitiveand are thus prime candidates for approaches based upon the control ofsynthesis of active steroids in peripheral target tissues. Thus, therate of formation of each sex steroid depends upon the activity of thespecific androgen- and estrogen-synthesizing enzymes in each cell ofeach tissue. Local hormone metabolism plays a key role in determiningtissue responsiveness to oestrogen. High capacity for inactivation ofoestrogens is associated with the presence of 17beta-HSD isozymes inepithelial cells. By inactivating oestrogens, NBHSD2 plays a role incancers, especially hormone-dependent cancers such as those stimulatedby androgens or estrogens, for example, colon, breast, prostate, ovarianand uterine cancer. In the colon, NBHSD2 plays a role as attenuator ofestradiol E2 bioavailability (estradiol (E2) stimulates the growth ofcolonic cancer cell lines), and possibly as modulators of colonic cellproliferation in the pathogenesis of colon cancer.

Also, bioavailibility of estradiol, one of the most potent human sexsteroid hormones of placental origin, is essential to the maintenance ofpregnancy, the timing of parturition, the maturation of many fetalorgans, and the preparation of the maternal reproductive system.

Several inhibitors of the functions of NBHSD2 have been characterized.These include: lindane which induces oxidative stress, progestins(promegestone, nomegestrol acetate, medrogestone) and tibolone and itsmetabolite which will provide a new possibility in the treatment ofbreast cancer, chalcones (naringenin chalcone and 4-hydroxychalcone),steroidal spirolactones inhibitors, isoflavones which have beensuggested to be anticarcinogenic, propylthiouracil (PTU) which is ananti-thyroid drug. Such inhibitors are useful tools to regulate thelevel of active estrogens, androgens and progesterone and can exertcancer-preventive effects. Alternatively, retinoic acids stimulate theexpression of NHBSD2 and may be involved in modulation of in situestrogen metabolism in both normal and neoplastic human endometrium.

An embodiment of the invention is directed to a composition comprising aNBHSD2 polypeptide sequence of SEQ ID NO:112.

A further embodiment of the invention is directed to a compositioncomprising a NBHSD2 polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:111 encoding a NBHSD2polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NBHSD2 polypeptidefragment having biological activity.

An embodiment of the invention provides for a method of screening testsubstances for modulators of NBHSD2 expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing NBHSD2 expression in the cell after exposure to the testsubstance to that of an unexposed control cell. NBHSD2 expression isdetermined by methods common to the art or included herein, by detectingNBHSD2 polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of NBHSD2 mRNA in eachsample by Northern blot, RTPCR, or another method common to the art. Theinvention provides for design and use of specific polynucleotide probesand primers, as discussed herein. An additional example comprises thesteps of: i) having two equivalent cultures of cells; ii) adding a testsubstance to one of the cultures and not the other; iii) harvesting bothcultures; iv) purifying the protein from each sample of cells; v)comparing the level of NBHSD2 polypeptides in each sample by Westernblot, immunohistochemistry, or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein.

Agents which modulate the expression or activity of the NBHSD2 of thesubject invention include, but are not limited to, antisenseoligonucleotides, ribozymes, drugs, and antibodies. These agents may bemade and used according to methods well known in the art. Also, theprotein of the invention, or biologically active fragments thereof, maybe used in screening assays for therapeutic compounds. A variety of drugscreening techniques may be employed. In this aspect of the invention,the protein or biologically active fragment thereof, may be free insolution, affixed to a solid support, recombinantly expressed on, orchemically attached to, a cell surface, or located intracellularly. Theformation of binding complexes, between the protein of the invention, orbiologically active fragments thereof, and the compound being tested,may then be measured. Another technique for drug screening which may beused provides for high throughput screening of compounds having suitablebinding affinity to the protein of the invention as described inpublished PCT application WO84/03564, and incorporated herein byreference in its entirety.

Another embodiment of the subject invention provides compositions andmethods of selectively modulating the activity of the protein of theinvention. Modulation of the NBHSD2 activity would allow for thesuccessful treatment and/or management of diseases or biochemicalabnormalities associated with the NBHSD2. Antagonists, able to reduce orinhibit the expression or the activity of the protein of the invention,would be useful in the treatment of diseases associated with decreasedestradiol and testosterone biosynthesis. For example, estradioldeficiency is an important pathogenetic factor in female osteoporosis.Also, antagonists of NBHSD2 provide methods of treating diseasesincluding, and not limited to, cancers, especially hormone-dependentcancers such as those stimulated by androgens or estrogens.Andogen-sentitive diseases, i.e. diseases whose onset or progress isaided by androgeneic activity, are known, included but are not limitedto prostate cancer, benign prostatic hyperplasia; acne, seborrhea,hirsutism, androgenic alopecia, precocious puberty, adrenal hyperplasiaand polycystic ovarian syndrome. Estrogen sensitive diseases, i.e.diseases whose onset or progress is aided by estrogenic activity,included but are not limited to breast cancer, endometriosis, leiomyoma,and precocious puberty.

Alternatively, the subject invention provides methods of treatingdiseases or disorders associated with decreased levels of the protein ofthe NBHSD2. Thus, agonists of NBHSD2 provide methods for treatingdiseases with increases in estradiol and testosterone levels.

In one embodiment, the subject method utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant nucleic acidsexpressing the NBHSD2 polypeptide or biologically active fragmentsthereof. The transformed cells may be viable or fixed. Drugs orcompounds which are candidates for the modulation of the NBHSD2, orbiologically active fragments thereof, are screened against suchtransformed cells in binding assays well known to those skilled in theart. Alternatively, assays such as those taught in Geysen H. N., WOApplication 84/03564, published on Sep. 13, 1984, and incorporatedherein by reference in its entirety, may be used to screen for peptidecompounds which demonstrate binding affinity for, or the ability tomodulate, the NBHSD2, or biologically active fragments thereof. Inanother embodiment, competitive drug screening assays using neutralizingantibodies specifically compete with a test compound for binding to theNBHSD2 protein of the invention, or biologically active fragmentsthereof.

Agents which stimulate or inhibit the activity of the protein of theinvention include but are not limited to agonist and antagonist drugsrespectively. These drugs can be obtained using any of a variety of drugscreening techniques as discussed above.

Antagonists of the NBHSD2 polypeptide encoded by SEQ ID NO:112 includeagents which decrease the levels of expressed mRNA encoding the proteinof SEQ ID NO:112. These include, but are not limited to, RNAi, one ormore ribozymes capable of digesting the protein of the invention mRNA,or antisense oligonucleotides capable of hybridizing to mRNA encodingthe NBHSD2 polypeptide of SEQ ID NO:112. Antisense oligonucleotides canbe administrated as DNA, as DNA entrapped in proteoliposomes containingviral envelope receptor proteins [Kanoda, Y. et al. (1989) Science 243:375, which disclosure is hereby incorporated by reference in itsentirety] or as part of a vector which can be expressed in the targetcell and provide antisense DNA or RNA. Vectors which are expressed inparticular cell types are known in the art. Alternatively, the DNA canbe injected along with a carrier. A carrier can be a protein such as acytokine, for example interleukin 2, or polylysine-glycoproteincarriers. Carrier proteins, vectors, and methods of making and usingpolylysine carrier systems are known in the art. Alternatively, nucleicacid encoding antisense molecules may be coated onto gold beads andintroduced into the skin with, for example, a gene gun [Ulmer, J. B. etal. (1993) Science 259:1745, which disclosure is hereby incorporated byreference in its entirety].

Antibodies, or other polypeptides, capable of reducing or inhibiting theactivity of NBHSD2 may be provided as in isolated and substantiallypurified form. Alternatively, antibodies or other polypeptides capableof inhibiting or reducing the activity of the protein of the invention,may be recombinantly expressed in the target cell to provide amodulating effect. In addition, compounds which inhibit or reduce theactivity of the protein of the subject invention may be incorporatedinto biodegradable polymers being implanted in the vicinity of wheredrug delivery is desired. For example, biodegradable polymers containingantagonists/agonists may be implanted to slowly release the compoundssystemically. Biodegradable polymers, and their use, are known to thoseof skill in the art (see, for example, Brem et al., J. Neurosurg.74:441-446(1991) which disclosure is hereby incorporated by reference inits entirety).

In one embodiment, methods of increasing the levels of NBHSD2 in tissuesor cell types may be practiced by utilizing nucleic acids encoding theprotein of the subject invention, or biologically active fragmentsthereof, to introduce biologically active polypeptide into targeted celltypes. Vectors useful in such methods are known to those skilled in theart as are methods of introducing such nucleic acids into targettissues. Preferred expression vectors include viral vectors, especiallyadenoviral and lentiviral vectors. For example, one of the methodsdescribed in Mulligan (Mulligan, Science, 260:926-32 (1993)), whichdisclosure is hereby incorporated by reference in its entirety, can beused.

In another embodiment, the invention provides methods and compositionsfor detecting the level of expression of the mRNA of the protein of theinvention. Quantification of mRNA levels of the NBHSD2 protein of theinvention may be useful for the diagnosis or prognosis of diseasesassociated with an altered expression of the protein of the invention.Assays for the detection and quantification of the mRNA of the proteinof the invention are well known in the art (see, for example, Maniatis,Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982), orCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (Eds),Wiley & Sons, Inc.).

Polynucleotides probes or primers for the detection of NBHSD2 cDNA canbe designed from the cDNA of SEQ ID NO:111. Methods for designing probesand primers are known in the art. In another embodiment, the subjectinvention provides diagnostic kits for the detection of NBHSD2 cDNA incells. The kit comprises a package having one or more containers ofoligonucleotide primers for detection of NBHSD2 cDNA in PCR assays orone or more containers of polynucleotide probes for the detection ofNBHSD2 cDNA by in situ hybridization or Northern analysis. Kits may,optionally, include containers of various reagents used in varioushybridization assays. The kit may also, optionally, contain one or moreof the following items: polymerization enzymes, buffers, instructions,controls, or detection labels. Kits may also, optionally, includecontainers of reagents mixed together in suitable proportions forperforming the hybridization assay methods in accordance with theinvention. Reagent containers preferably contain reagents in unitquantities that obviate measuring steps when performing the subjectmethods.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with an altered levelsof the protein of the invention. Diagnostic assays to detect the proteinof the invention may comprise a biopsy, in situ assay of cells fromorgan or tissue sections, or an aspirate of cells from a tumor or normaltissue. In addition, assays may be conducted upon cellular extracts fromorgans, tissues, cells, urine, or serum or blood or any other body fluidor extract.

Assays for the quantification of the NBHSD2 polypeptide of SEQ ID NO:112may be performed according to methods well known in the art. Typically,these assays comprise contacting the sample with a ligand of the proteinof the invention or an antibody (polyclonal or monoclonal) whichrecognizes the protein of the invention or a fragment thereof, anddetecting the complex formed between the protein of the inventionpresent in the sample and the ligand or antibody. Fragments of theligands and antibodies may also be used in the binding assays, providedthese fragments are capable of specifically interacting with the NBHSD2of the subject invention. Further, the ligands and antibodies which bindto the NBHSD2 of the invention may be labeled according to methods knownin the art. Labels which are useful in the subject invention include,but are not limited to, enzymes labels, radioisotopic labels,paramagnetic labels, and chemiluminescent labels. Typical techniques aredescribed by Kennedy, J. H., et al. (1976) Clin. Chim. Acta 70:1-31; andSchurs, A. H. et al. (1977) Clin. Chim. Acta 81: 140 which disclosure ishereby incorporated by reference in its entirety).

In another embodiment, the invention relates to compositions and methodsusing the proteins of the invention or fragment thereof to screen forcompounds that bind an NBHSD2 polypeptide or fragment thereof. In apreferred embodiment, the proteins of the invention or fragment thereofmay be used to identify and/or quantify substrates using any techniquesknown to those skilled in the art. To find substrates, the proteins ofthe invention, or fragment thereof, or derivative thereof, may be usedfor screening libraries of compounds in any of a variety of drugscreening techniques. The fragment employed in such screening may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenthe proteins of the invention, or fragment thereof, or derivativethereof, and the agent being tested, may be measured by methods wellknown to those skilled in the art, like, but not limited to, the BIAcore(Upsala, Sweden). Antagonists or inhibitors of the proteins of theinvention may be produced using methods which are generally known in theart, including the screening of libraries of pharmaceutical agents toidentify those which specifically bind the protein of the invention.Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of the invention.

In another embodiment, the present invention includes the use of NBHSD2polypeptides, or fragments having a desired biological activity to treator ameliorate a condition in an individual. For example, the conditionmay be deficiency of the sex steroid biosynthesis such ashormone-dependent disorders, or an abnormality in any of the functionsof the sex steroid metabolism. In such embodiments, AN NBHSD2polypeptide, or a fragment thereof, is administered to an individual inwhom it is desired to increase or decrease any of the activities ofNBHSD2 polypeptides. A NBHSD2 polypeptide or fragment thereof may beadministered directly to the individual or, alternatively, a nucleicacid encoding a NBHSD2 polypeptide or a fragment thereof may beadministered to the individual. Alternatively, an agent which increasesthe activity of NBHSD2 polypeptides may be administered to theindividual. Such agents may be identified by contacting a NBHSD2polypeptide or a cell or preparation containing NBHSD2 polypeptides witha test agent and assaying whether the test agent increases the activityof the protein. For example, the test agent may be a chemical compoundor a polypeptide or peptide. Alternatively, the activity of NBHSD2polypeptides may be decreased by administering an agent which interfereswith such activity to an individual. Agents which interfere with theactivity of NBHSD2 polypeptides may be identified by contacting A NBHSD2polypeptide or a cell or preparation containing NBHSD2 polypeptides witha test agent and assaying whether the test agent decreases the activityof the protein. Decreasing the activity of NHBSD2 would be useful forthe successful treatment and/or management of diseases or biochemicalabnormalities associated with decrease of oestradiol. For example, theagent may be a chemical compound, a polypeptide or peptide, an antibody,or a nucleic acid such as an antisense nucleic acid or a triplehelix-forming nucleic acid. Another embodiment of the invention relatesto composition and methods using polynucleotide sequences encoding theprotein of the invention or fragment thereof to establish transgenicmodel animals (D. melanogaster, M. musculus), by any method familiar tothose skilled in the art. By modulating in vivo the expression of thetransgene with drugs or modifier genes (activator or suppressor genes),animal models can be developed that mimic human hormone-dependentdisorders such as cancers. These animal models would thus allow theidentification of potential therapeutic agents for treatment of thedisorders. In addition, recombinant cell lines derived from thesetransgenic animals may be used for similar approaches ex vivo.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of SEQ ID NO:111 or fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations ordeletion. The microarray can be used to monitor the expression level oflarge numbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents (see for example: Chee, M. et al., Science,274:610-614 (1996) which disclosure is hereby incorporated by referencein its entirety). For example, deletion of genes NBHSD2 locus is afrequent target of deletion in human hepatocellular carcinoma.

Uses of Antibodies

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. An example of such use usingimmunoaffinity chromatography is given below. The antibodies of thepresent invention may be used either alone or in combination with othercompositions. For example, the antibodies have use in immunoassays forqualitatively and quantitatively measuring levels of antigen-bearingsubstances, including the polypeptides of the present invention, inbiological samples (See, e.g., Harlow et al., 1988). (Incorporated byreference in the entirety). The antibodies may also be used intherapeutic compositions for killing cells expressing the protein orreducing the levels of the protein in the body.

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies that disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies, which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies that bind the ligand and prevent binding of theligand to the receptor, as well as antibodies that bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies that activatethe receptor. These antibodies may act as agonists for either all orless than all of the biological activities affected by ligand-mediatedreceptor activation. The antibodies may be specified as agonists orantagonists for biological activities comprising specific activitiesdisclosed herein. The above antibody agonists can be made using methodsknown in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Denget al., (1998) Blood. 92(6):1981-1988; Chen et al., (1998), Cancer Res.58(16):3668-3678; Harrop et al., (1998), J. Immunol. 161(4): 1786-1794;Zhu, et al. (1998), Cancer Res. 58(15):3209-3214; Yoon, et (1998), J.Immunol. 160(7):3170-3179; Prat et al., (1998), J. Cell. Sci.111(Pt2):237-247; Pitard et al., (1997), J. Immunol. Methods.205(2):177-190; Liautard et al., (1997), Cytokine. 9(4):233-241; Carlsonet al., (1997), J. Biol. Chem. 272(17):11295-11301; Taryman, et al.,(1995), Neuron. 14(4):755-762; Muller et al., (1998), Structure.6(9):1153-1167; Bartunek et al., (1996), Cytokine. 8(1):14-20 (saidreferences incorporated by reference in their entireties).

As discussed above, antibodies of the polypeptides of the invention can,in turn, be utilized to generate anti-idiotypic antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art [See, e.g. Greenspan and Bona (1989), FASEB J.7(5):437-444 and Nissinoff, (1991), J. Immunol. 147(8): 2429-2438, whichdisclosures are hereby incorporated by reference in their entireties].For example, antibodies which bind to and competitively inhibitpolypeptide multimerization or binding of a polypeptide of the inventionto ligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization or binding domain and, as a consequence,bind to and neutralize polypeptide or its ligand. Such neutralizationanti-idiotypic antibodies can be used to bind a polypeptide of theinvention or to bind its ligands/receptors, and thereby block itsbiological activity.

Inmmunoaffinity Chromatography

Antibodies prepared as described herein are coupled to a support.Preferably, the antibodies are monoclonal antibodies, but polyclonalantibodies may also be used. The support may be any of those typicallyemployed in immunoaffinity chromatography, including Sepharose CL-4B(Pharmacia, Piscataway, N.J.), Sepharose CL-2B (Pharmacia, Piscataway,N.J.), Affi-gel 10 (Biorad, Richmond, Calif.), or glass beads.

The antibodies may be coupled to the support using any of the couplingreagents typically used in immunoaffinity chromatography, includingcyanogen bromide. After coupling the antibody to the support, thesupport is contacted with a sample which contains a target polypeptidewhose isolation, purification or enrichment is desired. The targetpolypeptide may be a polypeptide selected from the group consisting ofpolypeptide sequences of the Sequence Listing, those encoded by theclone inserts of the deposited clone pool, variants and fragmentsthereof, or a fusion protein comprising said selected polypeptide or afragment thereof.

Preferably, the sample is placed in contact with the support for asufficient amount of time and under appropriate conditions to allow atleast 50% of the target polypeptide to specifically bind to the antibodycoupled to the support.

Thereafter, the support is washed with an appropriate wash solution toremove polypeptides which have non-specifically adhered to the support.The wash solution may be any of those typically employed inimmunoaffinity chromatography, including PBS, Tris-lithium chloridebuffer (0.1M lysine base and 0.5M lithium chloride, pH 8.0),Tris-hydrochloride buffer (0.05M Tris-hydrochloride, pH 8.0), orTris/Triton/NaCl buffer (50 mM Tris.cl, pH 8.0 or 9.0, 0.1% TritonX-100, and 0.5MNaCl).

After washing, the specifically bound target polypeptide is eluted fromthe support using the high pH or low pH elution solutions typicallyemployed in immunoaffinity chromatography. In particular, the elutionsolutions may contain an eluant such as triethanolamine, diethylamine,calcium chloride, sodium thiocyanate, potasssium bromide, acetic acid,or glycine. In some embodiments, the elution solution may also contain adetergent such as Triton X-100 or octyl-beta-D-glucoside.

Expression of Genset Gene Products

Evaluation of Expression Levels and Patterns of GENSETPolypeptide-Encoding mRNAs

The spatial and temporal expression patterns of GENSETpolypeptide-encoding mRNAs, as well as their expression levels, may bedetermined as follows.

Expression levels and patterns of GENSET polypeptide-encoding mRNAs maybe analyzed by solution hybridization with long probes as described inInternational Patent Application No. WO 97/05277, the entire contents ofwhich are hereby incorporated by reference. Briefly, a GENSETpolynucleotide, or fragment thereof, corresponding to the gene encodingthe mRNA to be characterized is inserted at a cloning site immediatelydownstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter toproduce antisense RNA. Preferably, the GENSET polynucleotide is at leasta 100 nucleotides in length. The plasmid is linearized and transcribedin the presence of ribonucleotides comprising modified ribonucleotides(i.e. biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA ishybridized in solution with mRNA isolated from cells or tissues ofinterest. The hybridizations are performed under standard stringentconditions (40-50° C. for 16 hours in an 80% formamide, 0.4 M NaClbuffer, pH 7-8). The unhybridized probe is removed by digestion withribonucleases specific for single-stranded RNA (i.e. RNases CL3, T1, PhyM, U2 or A). The presence of the biotin-UTP modification enables captureof the hybrid on a microtitration plate coated with streptavidin. Thepresence of the DIG modification enables the hybrid to be detected andquantified by ELISA using an anti-DIG antibody coupled to alkalinephosphatase.

The GENSET polypeptide-encoding cDNAs, or fragments thereof, may also betagged with nucleotide sequences for the serial analysis of geneexpression (SAGE) as disclosed in UK Patent Application No. 2 305 241 A,the entire contents of which are incorporated by reference. In thismethod, cDNAs are prepared from a cell, tissue, organism or other sourceof nucleic acid for which it is desired to determine gene expressionpatterns. The resulting cDNAs are separated into two pools. The cDNAs ineach pool are cleaved with a first restriction endonuclease, called an“anchoring enzyme,” having a recognition site which is likely to bepresent at least once in most cDNAs. The fragments which contain the 5′or 3′ most region of the cleaved cDNA are isolated by binding to acapture medium such as streptavidin coated beads. A firstoligonucleotide linker having a first sequence for hybridization of anamplification primer and an internal restriction site for a “taggingendonuclease” is ligated to the digested cDNAs in the first pool.Digestion with the second endonuclease produces short “tag” fragmentsfrom the cDNAs. A second oligonucleotide having a second sequence forhybridization of an amplification primer and an internal restrictionsite is ligated to the digested cDNAs in the second pool. The cDNAfragments in the second pool are also digested with the “taggingendonuclease” to generate short “tag” fragments derived from the cDNAsin the second pool. The “tags” resulting from digestion of the first andsecond pools with the anchoring enzyme and the tagging endonuclease areligated to one another to produce “ditags.” In some embodiments, theditags are concatamerized to produce ligation products containing from 2to 200 ditags. The tag sequences are then determined and compared to thesequences of the GENSET polypeptide-encoding cDNAs to determine whichgenes are expressed in the cell, tissue, organism, or other source ofnucleic acids from which the tags were derived. In this way, theexpression pattern of a GENSET polypeptide-encoding gene in the cell,tissue, organism, or other source of nucleic acids is obtained.

Quantitative analysis of GENSET gene expression may also be performedusing arrays. For example, quantitative analysis of gene expression maybe performed with GENSET polynucleotides, or fragments thereof in acomplementary DNA microarray as described by Schena et al. (1995)Science 270:467470 and Schena et al. (1996), Proc Natl Acad Sci US A,.93(20):10614-10619 which disclosures are hereby incorporated byreference in their entireties. GENSET polypeptide-encoding cDNAs orfragments thereof are amplified by PCR and arrayed from 96-wellmicrotiter plates onto silylated microscope slides using high-speedrobotics. Printed arrays are incubated in a humid chamber to allowrehydration of the array elements and rinsed, once in 0.2% SDS for 1min, twice in water for 1 min and once for 5 min in sodium borohydridesolution. The arrays are submerged in water for 2 min at 95° C.,transferred into 0.2% SDS for 1 min, rinsed twice with water, air driedand stored in the dark at 25° C. Cell or tissue mRNA is isolated orcommercially obtained and probes are prepared by a single round ofreverse transcription. Probes are hybridized to 1 cm² microarrays undera 14×14 mm glass coverslip for 6-12 hours at 60° C. Arrays are washedfor 5 min at 25° C. in low stringency wash buffer (1×SSC/0.2% SDS), thenfor 10 min at room temperature in high stringency wash buffer(0.1×SSC/0.2% SDS). Arrays are scanned in 0.1×SSC using a fluorescencelaser scanning device fitted with a custom filter set. Accuratedifferential expression measurements are obtained by taking the averageof the ratios of two independent hybridizations.

Quantitative analysis of the expression of genes may also be performedwith GENSET polypeptide-encoding cDNAs or fragments thereof incomplementary DNA arrays as described by Pietu et al., (1996) GenomeResearch 6:492-503, which disclosure is hereby incorporated by referencein its entirety. The GENSET polynucleotides of the invention orfragments thereof are PCR amplified and spotted on membranes. Then,mRNAs originating from various tissues or cells are labeled withradioactive nucleotides. After hybridization and washing in controlledconditions, the hybridized mRNAs are detected by phospho-imaging orautoradiography. Duplicate experiments are performed and a quantitativeanalysis of differentially expressed mRNAs is then performed.

Alternatively, expression analysis of GENSET genes can be done throughhigh density nucleotide arrays as described by Lockhart et al., (1996)Nature Biotechnology 14: 1675-1680 and Sosnowski, et al., (1997) ProcNatl Acad Sci USA 94:1119-1123, which disclosures are herebyincorporated by reference in their entireties. Oligonucleotides of 15-50nucleotides corresponding to sequences of a GENSET polynucleotide orfragments thereof are synthesized directly on the chip (Lockhart et al.,supra) or synthesized and then addressed to the chip (Sosnowski et al.,supra). Preferably, the oligonucleotides are about 20 nucleotides inlength. cDNA probes labeled with an appropriate compound, such asbiotin, digoxigenin or fluorescent dye, are synthesized from theappropriate mRNA population and then randomly fragmented to an averagesize of 50 to 100 nucleotides. The said probes are then hybridized tothe chip. After washing as described in Lockhart et al., (supra) andapplication of different electric fields (Sosnowsky et al., supra), thedyes or labeling compounds are detected and quantified. Duplicatehybridizations are performed. Comparative analysis of the intensity ofthe signal originating from cDNA probes on the same targetoligonucleotide in different cDNA samples indicates a differentialexpression of the GENSET polypeptide-encoding mRNA.

Uses of GENSET Gene Expression Data

Once the expression levels and patterns of a GENSET polypeptide-encodingmRNA has been determined using any technique known to those skilled inthe art, in particular those described in the section entitled“Evaluation of Expression Levels and Patterns of GENSETpolypeptide-encoding mRNAs”, or using the instant disclosure, theseinformation may be used to design GENSET gene specific markers fordetection, identification, screening and diagnosis purposes as well asto design DNA constructs with an expression pattern similar to a GENSETgene expression pattern.

Detection of GENSET Polypeptide Expression and/or Biological Activity

The invention further relates to methods of detection of GENSETpolypeptide expression and/or biological activity in a biological sampleusing the polynucleotide and polypeptide sequences described herein.Such method scan be used, for example, as a screen for normal orabnormal GENSET polypeptide expression and/or biological activity and,thus, can be used diagnostically. The biological sample for use in themethods of the present invention includes a suitable sample from, forexample, a mammal, particularly a human.

Detection of GENSET Polypeptides

The invention further relates to methods of detection of GENSETpolypeptide or encoding polynucleotides in a sample using the sequencesdescribed herein and any techniques known to those skilled in the art.For example, a labeled polynucleotide probe having all or a functionalportion of the nucleotide sequence of a GENSET polypeptide-encodingpolynucleotide can be used in a method to detect a GENSETpolypeptide-encoding polynucleotide in a sample. In one embodiment, thesample is treated to render the polynucleotides in the sample availablefor hybridization to a polynucleotide probe, which can be DNA or RNA.The resulting treated sample is combined with a labeled polynucleotideprobe having all or a portion of the nucleotide sequence of the GENSETpolypeptide-encoding cDNA or genomic sequence, under conditionsappropriate for hybridization of complementary sequences to occur.Detection of hybridization of polynucleotides from the sample with thelabeled nucleic probe indicates the presence of GENSETpolypeptide-encoding polynucleotides in a sample. The presence of GENSETpolypeptide-encoding mRNA is indicative of GENSET polypeptide-encodinggene expression.

Consequently, the invention comprises methods for detecting the presenceof a polynucleotide comprising a nucleotide sequence selected from agroup consisting of the polynucleotide sequences of the SequenceListing, those of human cDNA clone inserts of the deposited clone pool,sequences fully complementary thereto, fragments and variants thereof ina sample. In a first embodiment, said method comprises the followingsteps of:

-   -   a) bringing into contact said sample and a nucleic acid probe or        a plurality of nucleic acid probes which hybridize to said        selected nucleotide sequence; and    -   b) detecting the hybrid complex formed between said probe or        said plurality of probes and said polynucleotide.

In a preferred embodiment of the above detection method, said nucleicacid probe or said plurality of nucleic acid probes is labeled with adetectable molecule. In another preferred embodiment of the abovedetection method, said nucleic acid probe or said plurality of nucleicacid probes has been immobilized on a substrate. In still anotherpreferred embodiment, said nucleic acid probe or said plurality ofnucleic acid probes has a sequence comprised in a sequence complementaryto said selected sequence.

In a second embodiment, said method comprises the steps of:

-   -   a) contacting said sample with amplification reaction reagents        comprising a pair of amplification primers located on either        side of the region of said nucleotide sequence to be amplified;    -   b) performing an amplification reaction to synthesize        amplification products containing said region of said selected        nucleotide sequence; and    -   c) detecting said amplification products.

In a preferred embodiment of the above detection method, when thepolynucleotide to be amplified is a RNA molecule, preliminary reversetranscription and synthesis of a second cDNA strand are necessary toprovide a DNA template to be amplified. In another preferred embodimentof the above detection method, the amplification product is detected byhybridization with a labeled probe having a sequence which iscomplementary to the amplified region. In still another preferredembodiment, at least one of said amplification primer has a sequencecomprised in said selected sequence or in the sequence complementary tosaid selected sequence.

Alternatively, a method of detecting GENSET polypeptide expression in atest sample can be accomplished using any product which binds to aGENSET polypeptide of the present invention or a portion of a GENSETpolypeptide. Such products may be antibodies, binding fragments ofantibodies, polypeptides able to bind specifically to GENSETpolypeptides or fragments thereof, including GENSET polypeptide agonistsand antagonists. Detection of specific binding to the antibody indicatesthe presence of a GENSET polypeptide in the sample (e.g., ELISA).

Consequently, the invention is also directed to a method for detectingspecifically the presence of a GENSET polypeptide according to theinvention in a biological sample, said method comprising the steps of:

-   -   a) bringing into contact said biological sample with a product        able to bind to a polypeptide of the invention or fragments        thereof;    -   b) allowing said product to bind to said polypeptide to form a        complex; and    -   c) detecting said complex.

In a preferred embodiment of the above detection method, the product isan antibody. In a more preferred embodiment, said antibody is labeledwith a detectable molecule. In another more preferred embodiment of theabove detection method, said antibody has been immobilized on asubstrate.

In addition, the invention also relates to methods of determiningwhether a GENSET gene product (e.g. a polynucleotide or polypeptide) ispresent or absent in a biological sample, said methods comprising thesteps of:

-   -   a) obtaining said biological sample from a human or non-human        animal, preferably a mammal;    -   b) contacting said biological sample with a product able to bind        to a GENSET polypeptide or encoding polynucleotide of the        invention; and    -   c) determining the presence or absence of said GENSET        polypeptide-encoding gene product in said biological sample.

The present invention also relates to kits that can be used in thedetection of GENSET polypeptide-encoding gene expression products. Thekit can comprise a compound that specifically binds a GENSET polypeptide(e.g. binding proteins, antibodies or binding fragments thereof (e.g.F(ab′)2 fragments) or a GENSET polypeptide-encoding mRNA (e.g. acomplementary probe or primer), for example, disposed within a containermeans. The kit can further comprise ancillary reagents, includingbuffers and the like.

Detection of GENSET Polypeptide Biological Activity

The invention further includes methods of detecting specifically aGENSET polypeptide biological activity, and to identify compoundscapable of modulating the activity of a GENSET polypeptide. Assessingthe GENSET polypeptide biological activity may be performed by thedetection of a change in any cellular property associated with theGENSET polypeptide, using a variety of techniques, including thosedescribed herein. To identify modulators of the polypeptides, a controlis preferably used. For example, a control sample includes all of thesame reagents but lacks the compound or agent being assessed; it istreated in the same manner as the test sample.

The present invention also relates to kits that can be used in thedetection of GENSET polypeptide biological activity. The kit cancomprise, e.g. substrates for GENSET polypeptides, GENSET-bindingcompounds, antibodies to GENSET polypeptides, etc., for example,disposed within a container means. The kit can further compriseancillary reagents, including buffers and the like.

Identification of a Specific Context of GENSET Polypeptide-Encoding GeneExpression

When the expression pattern of a GENSET polypeptide-encoding mRNA showsthat a GENSET polypeptide-encoding gene is specifically expressed in agiven context, probes and primers specific for this gene as well asantibodies binding to the GENSET polypeptide-encoding polynucleotide maythen be used as markers for the specific context. Examples of specificcontexts are: specific expression in a given tissue/cell or tissue/celltype, expression at a given stage of development of a process such asembryo development or disease development, or specific expression in agiven organelle. Such primers, probes, and antibodies are usefulcommercially to identify tissues/cells/organelles of unknown origin, forexample, forensic samples, differentiated tumor tissue that hasmetastasized to foreign bodily sites, or to differentiate differenttissue types in a tissue cross-section using any technique known tothose skilled in the art including in situ PCR or immunochemistry forexample.

For example, the cDNAs and proteins of the sequence listing andfragments thereof, may be used to distinguish human tissues/cells fromnon-human tissues/cells and to distinguish between humantissues/cells/organelles that do and do not express the polynucleotidescomprising the cDNAs. By knowing the expression pattern of a givenGENSET polypeptide, either through routine experimentation or by usingthe instant disclosure, the polynucleotides and polypeptides of thepresent invention may be used in methods of determining the identity ofan unknown tissue/cell sample/organelle. As part of determining theidentity of an unknown tissue/cell sample/organelle, the polynucleotidesand polypeptides of the present invention may be used to determine whatthe unknown tissue/cell sample is and what the unknown sample is not.For example, if a cDNA is expressed in a particular tissue/celltype/organelle, and the unknown tissue/cell sample/organelle does notexpress the cDNA, it may be inferred that the unknown tissue/cells areeither not human or not the same human tissue/cell type/organelle asthat which expresses the cDNA. Determination of tissue/cell/organelleidentity is based on methods that detect the presence or absence of themRNA (or corresponding cDNA) in a tissue/cell sample using methods wellknown in the art (e.g., hybridization, PCR based methods, immunoassays,immunochemistry, ELISA). Examples of such techniques are described inmore detail below. Therefore, the invention encompasses uses of thepolynucleotides and polypeptides of the invention as tissue markers.Consequently, the present invention encompasses methods ofidentification of a tissue/cell type/subcellular compartment, whereinsaid method includes the steps of:

-   -   a) contacting a biological sample which identity is to be        assayed with a product able to bind a GENSET gene product; and    -   b) determining whether a GENSET gene product is expressed in        said biological sample.

Products that are able to bind specifically to a GENSET gene product,namely a GENSET polypeptide or a GENSET polypeptide-encoding mRNA,include GENSET polypeptide binding proteins, antibodies or bindingfragments thereof (e.g. F(ab′)2 fragments), as well as GENSETpolynucleotide complementary probes and primers.

Step b) may be performed using any detection method known to thoseskilled in the art including those disclosed herein, especially in thesection entitled “Detection of GENSET polypeptide expression and/orbiological activity”.

Identification of Tissue Types or Cell Species by Means of LabeledTissue Specific Antibodies

Identification of specific tissues is accomplished by the visualizationof tissue specific antigens by means of antibody preparations which areconjugated, directly (e.g., green fluorescent protein) or indirectly toa detectable marker. Selected labeled antibody species bind to theirspecific antigen binding partner in tissue sections, cell suspensions,or in extracts of soluble proteins from a tissue sample to provide apattern for qualitative or semi-qualitative interpretation.

A. Immunohistochemical Techniques

Purified, high-titer antibodies, prepared as described above, areconjugated to a detectable marker, as described, for example, byFudenberg, (1980) Chap. 26 in: Basic 503 Clinical Immunology, 3rd Ed.Lange, Los Altos, Calif. or Rose et al., (1980) Chap. 12 in: Methods inImmunodiagnosis, 2d Ed. John Wiley 503 Sons, New York, which disclosuresare hereby incorporated by reference in their entireties.

A fluorescent marker, either fluorescein or rhodamine, is preferred, butantibodies can also be labeled with an enzyme that supports a colorproducing reaction with a substrate, such as horseradish peroxidase.Markers can be added to tissue-bound antibody in a second step, asdescribed below. Alternatively, the specific anti-tissue antibodies canbe labeled with ferritin or other electron dense particles, andlocalization of the ferritin coupled antigen-antibody complexes achievedby means of an electron microscope. In yet another approach, theantibodies are radiolabeled, with, for example ¹²⁵I, and detected byoverlaying the antibody treated preparation with photographic emulsion.Preparations to carry out the procedures can comprise monoclonal orpolyclonal antibodies to a single protein or peptide identified asspecific to a tissue type, for example, brain tissue, or antibodypreparations to several antigenically distinct tissue specific antigenscan be used in panels, independently or in mixtures, as required. Tissuesections and cell suspensions are prepared for immunohistochemicalexamination according to common histological techniques. Multiplecryostat sections (about 4 um, unfixed) of the unknown tissue and knowncontrol, are mounted and each slide covered with different dilutions ofthe antibody preparation. Sections of known and unknown tissues shouldalso be treated with preparations to provide a positive control, anegative control, for example, pre-immune sera, and a control fornon-specific staining, for example, buffer. Treated sections areincubated in a humid chamber for 30 min at room temperature, rinsed,then washed in buffer for 30-45 min. Excess fluid is blotted away, andthe marker developed. If the tissue specific antibody was not labeled inthe first incubation, it can be labeled at this time in a secondantibody-antibody reaction, for example, by adding fluorescein- orenzyme-conjugated antibody against the immunoglobulin class of theantiserum-producing species, for example, fluorescein labeled antibodyto mouse IgG. Such labeled sera are commercially available. The antigenfound in the tissues by the above procedure can be quantified bymeasuring the intensity of color or fluorescence on the tissue section,and calibrating that signal using appropriate standards.

B. Identification of Tissue Specific Soluble Proteins

The visualization of tissue specific proteins and identification ofunknown tissues from that procedure is carried out using the labeledantibody reagents and detection strategy as described forimmunohistochemistry; however the sample is prepared according to anelectrophoretic technique to distribute the proteins extracted from thetissue in an orderly array on the basis of molecular weight fordetection. For example, Western Blot Analysis, see, e.g., Davis et al.,Basic Methods in Molecular Biology, ed., Elsevier Press, NY (1986),Section 19-3.

In either procedure A or B, a detectable label can be attached to theprimary tissue antigen-primary antibody complex according to variousstrategies and permutations thereof. In a straightforward approach, theprimary specific antibody can be labeled; alternatively, the unlabeledcomplex can be bound by a labeled secondary anti-IgG antibody. In otherapproaches, either the primary or secondary antibody is conjugated to abiotin molecule, which can, in a subsequent step, bind an avidinconjugated marker. According to yet another strategy, enzyme labeled orradioactive protein A, which has the property of binding to any IgG, isbound in a final step to either the primary or secondary antibody. Thevisualization of tissue specific antigen binding at levels above thoseseen in control tissues to one or more tissue specific antibodies,prepared from the gene sequences identified from cDNA sequences, canidentify tissues of unknown origin, for example, forensic samples, ordifferentiated tumor tissue that has metastasized to foreign bodilysites.

Screening and Diagnosis of Abnormal GENSET Polypeptide Expression and/orBiological Activity

Moreover, antibodies and/or primers specific for GENSET polypeptideexpression may also be used to identify abnormal GENSET polypeptideexpression and/or biological activity, and subsequently to screen and/ordiagnose disorders associated with abnormal GENSET polypeptideexpression. For example, a particular disease may result from lack ofexpression, over expression, or under expression of a GENSETpolypeptide-encoding mRNA. By comparing mRNA expression patterns andquantities in samples taken from healthy individuals with those fromindividuals suffering from a particular disorder, genes responsible forthis disorder may be identified. Primers, probes and antibodies specificfor this GENSET polypeptide may then be used to elaborate kits ofscreening and diagnosis for a disorder in which the gene of interest isspecifically expressed or in which its expression is specificallydysregulated, i.e. underexpressed or overexpressed.

Screening for Specific Disorders

The present invention also relates to methods and uses of GENSETpolypeptides for identifying individuals having elevated or reducedlevels of GENSET polypeptides, which individuals are likely to benefitfrom therapies to suppress or enhance GENSET polypeptide-encoding geneexpression, respectively. One example of such methods and uses comprisesthe steps of:

-   -   a) obtaining from a mammal a biological sample;    -   b) detecting the presence in said sample of a GENSET        polypeptide-encoding gene product (mRNA or protein);    -   c) comparing the amount of said GENSET polypeptide-encoding gene        product present in said sample with that of a control sample;        and    -   d) determining whether said human or non-human mammal has a        reduced or elevated level of GENSET gene expression compared to        the control sample.

A biological sample from a subject affected by, or at risk ofdeveloping, any disease or condition associated with a GENSETpolypeptide can be screened for the presence of increased or decreasedlevels of GENSET gene product, relative to a normal population (standardor control), with an increased or decreased level of the GENSETpolypeptide relative to the normal population being indicative ofpredisposition to or a present indication of the disease or condition,or any sympton associated with the disease or condition. Suchindividuals would be candidates for therapies, e.g., treatment withpharmaceutical compositions comprising the GENSET polypeptide, apolynucleotide encoding the GENSET polypeptide, or any other compoundthat affects the expression or activity of the GENSET polypeptide.Generally, the identification of elevated levels of the GENSETpolypeptide in a patient would be indicative of an individual that wouldbenefit from treatment with agents that suppress GENSET polypeptideexpression or activity, and the identification of low levels of theGENSET polypeptide in a patient would be indicative of an individualthat would benefit from agents that induce GENSET expression oractivity.

Biological samples suitable for use in this method include anybiological fluids, including, but not limited to, blood, saliva, milk,and urine. Tissue samples (e.g. biopsies) can also be used in the methodof the invention, including samples derived from any tissue associatedwith GENSET gene expression as determined by any method common to theart such as those described herein. Cell cultures or cell extractsderived, for example, from tissue biopsies can also be used. Thedetection step of the present method can be performed using standardprotocols for protein/mRNA detection. Examples of suitable protocolsinclude Northern blot analysis, immunoassays (e.g. RIA, Western blots,immunohistochemical analyses), and PCR.

Thus, the present invention further relates to methods and uses ofGENSET polypeptides for identifying individuals or non-human animals atincreased risk for developing, or present state of having, certaindiseases/disorders associated with abnormal GENSET polypeptideexpression or biological activity. One example of such methods comprisesthe steps of:

-   -   a) obtaining from a human or non-human mammal a biological        sample;    -   b) detecting the presence in said sample of a GENSET gene        product (mRNA or protein);    -   c) comparing the amount of said GENSET gene product present in        said sample with that of a control sample; and    -   d) determining whether said human or non-human mammal is at        increased risk for developing, or present state of having, a        diseases or disorder.

In preferred embodiments, the biological sample is taken from animalspresenting any symptom associated with any disease or conditionassociated with a GENSET gene product. In accordance with this method,the presence in the sample of altered (e.g. increased or decreased)levels of the GENSET product indicates that the subject is predisposedto the disease or condition. Biological samples suitable for use in thismethod include biological fluids including, but not limited to, blood,saliva, milk, and urine. Tissue samples (e.g. biopsies) can also be usedin the method of the invention. Cell cultures or cell extracts derived,for example, from tissue biopsies can also be used.

The diagnostic methodologies described herein are applicable to bothhumans and non-human mammals.

Detection of GENSET Gene Mutations

The invention also encompasses methods and uses of GENSETpolynucleotides to detect mutations in GENSET polynucleotides of theinvention. Such methods may advantageously be used to detect mutationsoccurring in GENSET genes and preferably in their regulatory regions.When the mutation was proven to be associated with a disease, thedetection of such mutations may be used for screening and diagnosispurposes.

In one embodiment of the oligonucleotide arrays of the invention, anoligonucleotide probe matrix may advantageously be used to detectmutations occurring in GENSET genes and preferably in their regulatoryregions. For this particular purpose, probes are specifically designedto have a nucleotide sequence allowing their hybridization to the genesthat carry known mutations (either by deletion, insertion orsubstitution of one or several nucleotides). By known mutations, it ismeant, mutations on the GENSET genes that have been identifiedaccording, for example to the technique used by Huang et al., (1996)Cancer Res 56(5):1137-1141 or Samson et al., (1996) Nature,382(6593):722-725, which disclosures are hereby incorporated byreference in their entireties.

Another technique that is used to detect mutations in GENSET genes isthe use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of a GENSET genomic DNA or cDNA. Thus, anarray consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity of the targetsequence with the wild gene sequence, measure its amount, and detectdifferences between the target sequence and the reference wild genesequence of the GENSET gene. In one such design, termed 4L tiled array,is implemented a set of four probes (A, C, G, T), preferably15-nucleotide oligomers. In each set of four probes, the perfectcomplement will hybridize more strongly than mismatched probes.Consequently, a nucleic acid target of length L is scanned for mutationswith a tiled array containing 4L probes, the whole probe set containingall the possible mutations in the known wild reference sequence. Thehybridization signals of the 15-mer probe set tiled array are perturbedby a single base change in the target sequence. As a consequence, thereis a characteristic loss of signal or a “footprint” for the probesflanking a mutation position. This technique was described by Chee etal., (1996) Science. 274:610-614, which disclosure is herebyincorporated by reference in its entirety.

Construction of DNA Constructs with a GENSET Gene Expression Pattern

In addition, characterization of the spatial and temporal expressionpatterns and expression levels of GENSET polypeptide-encoding mRNAs isalso useful for constructing expression vectors capable of producing adesired level of gene product in a desired spatial or temporal manner,as discussed below.

DNA Constructs that Direct Temporal and Spatial GENSET Gene Expressionin Recombinant Cell Hosts and in Transgenic Animals.

In order to study the physiological and phenotypic consequences of alack of synthesis of a GENSET polypeptide, both at the cellular leveland at the multi cellular organism level, the invention also encompassesDNA constructs and recombinant vectors enabling a conditional expressionof a specific allele of a GENSET polypeptide-encoding genomic sequenceor cDNA and also of a copy of this genomic sequence or cDNA harboringsubstitutions, deletions, or additions of one or more bases as regardsto a polynucleotide of the present regulatory sequence, but preferablyin the 5′-regulatory sequence or in an exon of the GENSETpolypeptide-encoding genomic sequence or within the GENSETpolypeptide-encoding cDNA.

A first preferred DNA construct is based on the tetracycline resistanceoperon tet from E. coli transposon Tn10 for controlling the GENSET geneexpression, such as described by Gossen et al., (1992) Proc. Natl. Acad.Sci. USA. 89:5547-5551; Gossen et al, (1995) Science 268:1766-1769; andFurth P. A. et al. (1994) Proc. Natl. Acad. Sci USA. 91:9302-9306, whichdisclosures are hereby incorporated by reference in their entireties.Such a DNA construct contains seven tet operator sequences from Tn10(tetop) that are fused to either a minimal promoter or a 5′-regulatorysequence of the GENSET gene, said minimal promoter or said GENSETpolynucleotide regulatory sequence being operably linked to apolynucleotide of interest that codes either for a sense or an antisenseoligonucleotide or for a polypeptide, including a GENSET polypeptide, ora peptide fragment thereof. This DNA construct is functional as aconditional expression system for the nucleotide sequence of interestwhen the same cell also comprises a nucleotide sequence coding foreither the wild type (tTA) or the mutant (rTA) repressor fused to theactivating domain of viral protein VP16 of herpes simplex virus, placedunder the control of a promoter, such as the HCMVIE1 enhancer/promoteror the MMTV-LTR. Indeed, a preferred DNA construct of the inventioncomprise both the polynucleotide containing the tet operator sequencesand the polynucleotide containing a sequence coding for the tTA or therTA repressor. In a specific embodiment, the conditional expression DNAconstruct contains the sequence encoding the mutant tetracyclinerepressor rT A, the expression of the polynucleotide of interest issilent in the absence of tetracycline and induced in its presence.

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A second preferred DNA construct will comprise, from 5′-end to 3′-end:(a) a first nucleotide sequence that is found in the GENSETpolypeptide-encoding genomic sequence; (b) a nucleotide sequencecomprising a positive selection marker, such as the marker for neomycinresistance (neo); and (c) a second nucleotide sequence that is found inthe GENSET polypeptide-encoding genomic sequence, and is located on thegenome downstream the first GENSET polypeptide-encoding nucleotidesequence (a).

In a preferred embodiment, this DNA construct also comprises a negativeselection marker located upstream of the nucleotide sequence (a) ordownstream from the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene [Thomas et al.(1986), Cell. 44:419-428], the hygromycine beta gene [Te Riele et al.(1990), Nature. 348:649-651], the hprt gene [Van der Lugt et al. (1991),Gene. 105:263-267; Reid et al., (1990) Proc. Natl. Acad. Sci. U.S.A.87:4299-4303] or the Diphteria toxin A fragment (Dt-A) gene [Nada etal., (1993) Cell 73:1125-1135; Yagi, T., et al. (1990), Proc. Natl.Acad. Sci. U.S.A. 87:9918-992], which disclosures are herebyincorporated by reference in their entireties. Preferably, the positiveselection marker is located within a GENSET exon sequence so as tointerrupt the sequence encoding a GENSET polypeptide. These replacementvectors are described, for example, by Thomas et al(1986; 1987), Mansouret al. (1988) and Koller et al., (1992) Annu. Rev. Immunol. 10:705-730.

The first and second nucleotide sequences (a) and (c) may beindifferently located within a GENSET polypeptide-encoding regulatorysequence, an intronic sequence, an exon sequence or a sequencecontaining both regulatory and/or intronic and/or exon sequences. Thesize of the nucleotide sequences (a) and (c) ranges from 1 to 50 kb,preferably from 1 to 10 kb, more preferably from 2 to 6 kb and mostpreferably from 2 to 4 kb.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.

These new DNA constructs make use of the site specific recombinationsystem of the P1 phage. The P1 phage possesses a recombinase called Crewhich interacts specifically with a 34 base pairs loxP site. The loxPsite is composed of two palindromic sequences of 13 bp separated by a 8bp conserved sequence [Hoess et al., (1986) Nucleic Acids Res.14:2287-2300], which disclosure is hereby incorporated by reference inits entirety. The recombination by the Cre enzyme between two loxP siteshaving an identical orientation leads to the deletion of the DNAfragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu H. et al., (1993) Cell73:1155-1164 and Gu H. et al., (1994) Science 265:103-106, whichdisclosures are hereby incorporated by reference in their entireties.Briefly, a nucleotide sequence of interest to be inserted in a targetedlocation of the genome harbors at least two loxP sites in the sameorientation and located at the respective ends of a nucleotide sequenceto be excised from the recombinant genome. The excision event requiresthe presence of the recombinase (Cre) enzyme within the nucleus of therecombinant cell host. The recombinase enzyme may be brought at thedesired time either by (a) incubating the recombinant cell hosts in aculture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as described by Araki et al.,(1995) Proc. Natl. Acad. Sci. USA. 92(1):1604, which disclosure ishereby incorporated by reference in its entirety, or by lipofection ofthe enzyme into the cells, such as described by Baubonis et al (1993)Nucleic Acids Res. 21(9):2025-9), which disclosure is herebyincorporated by reference in its entirety; (b) transfecting the cellhost with a vector comprising the Cre coding sequence operably linked toa promoter functional in the recombinant cell host, which promoter beingoptionally inducible, said vector being introduced in the recombinantcell host, such as described by Gu et al. (1993) and Sauer et al.,(1988) Proc. Natl. Acad. Sci. U.S.A. 85:5166-5170, which disclosures arehereby incorporated by reference in their entireties; (c) introducing inthe genome of the cell host a polynucleotide comprising the Cre codingsequence operably linked to a promoter functional in the recombinantcell host, which promoter is optionally inducible, and saidpolynucleotide being inserted in the genome of the cell host either by arandom insertion event or an homologous recombination event, such asdescribed by Gu et al. (1994).

In a specific embodiment, the vector containing the sequence to beinserted in the GENSET gene by homologous recombination is constructedin such a way that selectable markers are flanked by loxP sites of thesame orientation, it is possible, by treatment by the Cre enzyme, toeliminate the selectable markers while leaving the GENSET sequences ofinterest that have been inserted by an homologous recombination event.Again, two selectable markers are needed: a positive selection marker toselect for the recombination event and a negative selection marker toselect for the homologous recombination event. Vectors and methods usingthe Cre-loxP system are described by Zou, et al, (1994) Curr. Biol.4:1099-1103, which disclosure is hereby incorporated by reference in itsentirety.

Thus, a third preferred DNA construct of the invention comprises, from5′-end to 3′-end: (a) a first nucleotide sequence that is comprised inthe GENSET genomic sequence; (b) a nucleotide sequence comprising apolynucleotide encoding a positive selection marker, said nucleotidesequence comprising additionally two sequences defining a siterecognized by a recombinase, such as a loxP site, the two sites beingplaced in the same orientation; and (c) a second nucleotide sequencethat is comprised in the GENSET genomic sequence, and is located on thegenome downstream of the first GENSET nucleotide sequence (a).

The sequences defining a site recognized by a recombinase, such as aloxP site, are preferably located within the nucleotide sequence (b) atsuitable locations bordering the nucleotide sequence for which theconditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA constructdescribed above, the excision of the polynucleotide fragment bordered bythe two sites recognized by a recombinase, preferably two loxP sites, isperformed at a desired time, due to the presence within the genome ofthe recombinant host cell of a sequence encoding the Cre enzyme operablylinked to a promoter sequence, preferably an inducible promoter, morepreferably a tissue-specific promoter sequence and most preferably apromoter sequence which is both inducible and tissue-specific, such asdescribed by Gu et al. (1994).

The presence of the Cre enzyme within the genome of the recombinant cellhost may result from the breeding of two transgenic animals, the firsttransgenic animal bearing the GENSET-derived sequence of interestcontaining the loxP sites as described above and the second transgenicanimal bearing the Cre coding sequence operably linked to a suitablepromoter sequence, such as described by Gu et al. (1994).

Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton and Graham,(1995), J. Virol., 69: 4600-4606 and Kanegae et al., (1995) Nucl. AcidsRes. 23:3816-3821, which disclosures are hereby incorporated byreference in their entireties.

The DNA constructs described above may be used to introduce a desirednucleotide sequence of the invention, preferably a GENSET genomicsequence or a GENSET cDNA sequence, and most preferably an altered copyof a GENSET genomic or cDNA sequence, within a predetermined location ofthe targeted genome, leading either to the generation of an altered copyof a targeted gene (knock-out homologous recombination) or to thereplacement of a copy of the targeted gene by another copy sufficientlyhomologous to allow an homologous recombination event to occur (knock-inhomologous recombination).

Modifying Genset Polypoptide Expression and/or Biological Activity

Modifying endogenous GENSET expression and/or biological activity isexpressly contemplated by the present invention.

Screening for Compounds that Modulate GENSET Expression and/orBiological Activity

The present invention further relates to compounds able to modulateGENSET expression and/or biological activity and methods to use thesecompounds. Such compounds may interact with the regulatory sequences ofGENSET genes or they may interact with GENSET polypeptides directly orindirectly.

Compounds Interacting with GENSET Regulatory Sequences

The present invention also concerns a method for screening substances ormolecules that are able to interact with the regulatory sequences of aGENSET gene, such as for example promoter or enhancer sequences inuntranscribed regions of the genomic DNA, as determined using anytechniques known to those skilled in the art including those describedin the section entitled “Identification of Promoters in Cloned UpstreamSequences, or such as regulatory sequences located in untranslatedregions of GENSET mRNA.

Sequences within untranscribed or untranslated regions ofpolynucleotides of the invention may be identified by comparison todatabases containing known regulatory sequence such as transcriptionstart sites, transcription factor binding sites, promoter sequences,enhancer sequences, 5′ UTR and 3′ UTR elements [Pesole et al., (2000)Nucleic Acids Res, 28(1):193-196;http://igs-server.cnrs-mrs.fr/˜gauthere/UTR/index.html]. Alternatively,the regulatory sequences of interest may be identified throughconventional mutagenesis or deletion analyses of reporter plasmidsusing, for instance, techniques described in the section entitled“Identification of Promoters in Cloned Upstream Sequences”.

Following the identification of potential GENSET regulatory sequences,proteins which interact with these regulatory sequences may beidentified as described below.

Gel retardation assays may be performed independently in order to screencandidate molecules that are able to interact with the regulatorysequences of the GENSET gene, such as described by Fried and Crothers,(1981) Nucleic Acids Res. 9:6505-6525, Garner and Revzin, (1981) NucleicAcids Res 9:3047-3060 and Dent and Latchman (1993) The DNA mobilityshift assay. In: Transcription Factors: A Practical Approach (LatchmanDS, ed.) pp 1-26. Oxford: IRL Press, the teachings of these publicationsbeing herein incorporated by reference. These techniques are based onthe principle according to which a DNA or mRNA fragment which is boundto a protein migrates slower than the same unbound DNA or mRNA fragment.Briefly, the target nucleotide sequence is labeled. Then the labeledtarget nucleotide sequence is brought into contact with either a totalnuclear extract from cells containing regulation factors, or withdifferent candidate molecules to be tested. The interaction between thetarget regulatory sequence of the GENSET gene and the candidate moleculeor the regulation factor is detected after gel or capillaryelectrophoresis through a retardation in the migration.

Nucleic acids encoding proteins which are able to interact with thepromoter sequence of the GENSET gene, more particularly thepolynucleotides of the 5′ and 3′ regulatory region or a fragment orvariant thereof, may be identified by using a one-hybrid system, such asthat described in the booklet enclosed in the Matchmaker One-HybridSystem kit from Clontech (Catalog Ref. no. K1603-1, the technicalteachings of which are herein incorporated by reference).

Ligands Interacting with GENSET Polypeptides

For the purpose of the present invention, a ligand means a molecule,such as a protein, a peptide, an antibody or any synthetic chemicalcompound capable of binding to a GENSET protein or one of its fragmentsor variants or to modulate the expression of the polynucleotide codingfor GENSET or a fragment or variant thereof.

In the ligand screening method according to the present invention, abiological sample or a defined molecule to be tested as a putativeligand of a GENSET protein is brought into contact with thecorresponding purified GENSET protein, for example the correspondingpurified recombinant GENSET protein produced by a recombinant cell hostas described herein, in order to form a complex between this protein andthe putative ligand molecule to be tested.

As an illustrative example, to study the interaction of a polypeptide ofthe invention, with drugs or small molecules, such as moleculesgenerated through combinatorial chemistry approaches, the microdialysiscoupled to HPLC method described by Wang, et al. (1997),Chromatographia, 44: 205-208 or the affinity capillary electrophoresismethod described by Bush et al., (1997), J. Chromatogr., 777: 311-328,the disclosures of which are incorporated by reference, can be used.

In further methods, peptides, drugs, fatty acids, lipoproteins, or smallmolecules which interact with a may be identified using assays known inthe art. For example, the molecule to be tested for binding is labeledwith a detectable label, such as a fluorescent, radioactive, orenzymatic tag and placed in contact with immobilized GENSET protein, ora fragment thereof under conditions which permit specific binding tooccur. After removal of non-specifically bound molecules, boundmolecules are detected using appropriate means.

Various candidate substances or molecules can be assayed for interactionwith a GENSET polypeptide. These substances or molecules include,without being limited to, natural or synthetic organic compounds ormolecules of biological origin such as polypeptides. When the candidatesubstance or molecule comprises a polypeptide, this polypeptide may bethe resulting expression product of a phage clone belonging to aphage-based random peptide library, or alternatively the polypeptide maybe the resulting expression product of a cDNA library cloned in a vectorsuitable for performing a two-hybrid screening assay.

A. Candidate Ligands Obtained from Random Peptide Libraries

In a particular embodiment of the screening method, the putative ligandis the expression product of a DNA insert contained in a phage vector[Parmley and Smith, (1988) Gene 73:305-318]. Specifically, randompeptide phages libraries are used. The random DNA inserts encode forpeptides of 8 to 20 amino acids in length [Oldenburg et al., (1992),Proc. Natl. Acad. Sci. USA 89:5393-5397; Valadon et al., (1996), J. Mol.Biol., 261:11-22; Lucas (1994), In: Development and Clinical Uses ofHaempophilus b Conjugate; Westerink (1995), Proc. Natl. Acad. Sci USA.,92:4021-4025; Felici, (1991), J. Mol. Biol., 222:301-310], whichdisclosures are hereby incorporated by reference in their entireties.According to this particular embodiment, the recombinant phagesexpressing a protein that binds to an immobilized GENSET protein isretained and the complex formed between the GENSET protein and therecombinant phage may be subsequently immunoprecipitated by a polyclonalor a monoclonal antibody directed against the GENSET protein.

Once the ligand library in recombinant phages has been constructed, thephage population is brought into contact with the immobilized GENSETprotein. Then the preparation of complexes is washed in order to removethe non-specifically bound recombinant phages. The phages that bindspecifically to the GENSET protein are then eluted by a buffer (acid pH)or immunoprecipitated by the monoclonal antibody produced by thehybridoma anti-GENSET, and this phage population is subsequentlyamplified by an over-infection of bacteria (for example E. coli). Theselection step may be repeated several times, preferably 2-4 times, inorder to select the more specific recombinant phage clones. The laststep comprises characterizing the peptide produced by the selectedrecombinant phage clones either by expression in infected bacteria andisolation, expressing the phage insert in another host-vector system, orsequencing the insert contained in the selected recombinant phages.

B. Candidate Ligands Obtained by Competition Experiments.

Alternatively, peptides, drugs or small molecules which bind topolypeptide of the present invention may be identified in competitionexperiments. In such assays, the GENSET protein, or a fragment thereof,is immobilized to a surface, such as a plastic plate. Increasing amountsof the peptides, drugs or small molecules are placed in contact with theimmobilized GENSET protein, or a fragment thereof, in the presence of adetectable labeled known GENSET protein ligand. For example, the GENSETligand may be detectably labeled with a fluorescent, radioactive, orenzymatic tag. The ability of the test molecule to bind the GENSETprotein, or a fragment thereof, is determined by measuring the amount ofdetectably labeled known ligand bound in the presence of the testmolecule. A decrease in the amount of known ligand bound to the GENSETprotein, or a fragment thereof, when the test molecule is presentindicated that the test molecule is able to bind to the GENSET protein,or a fragment thereof.

C. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with a polypeptide of thepresent invention, can also be found using affinity columns whichcontain the GENSET protein, or a fragment thereof. The GENSET protein,or a fragment thereof, may be attached to the column using conventionaltechniques including chemical coupling to a suitable column matrix suchas agarose, Affi Gel®, or other matrices familiar to those of skill inart. In some embodiments of this method, the affinity column containschimeric proteins in which the GENSET protein, or a fragment thereof, isfused to glutathion S transferase (GST). A mixture of cellular proteinsor pool of expressed proteins as described above is applied to theaffinity column. Proteins or other molecules interacting with the GENSETprotein, or a fragment thereof, attached to the column can then beisolated and analyzed on 2-D electrophoresis gel as described inRamunsen et al, (1997), Electrophoresis, 18: 588-598, the disclosure ofwhich is incorporated by reference. Alternatively, the proteins retainedon the affinity column can be purified by electrophoresis based methodsand sequenced. The same method can be used to isolate antibodies, toscreen phage display products, or to screen phage display humanantibodies.

D. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with a polypeptide of the present invention, canalso be screened by using an Optical Biosensor as described in Edwardsand Leatherbarrow, (1997) Analytical Biochemistry, 246, 1-6 and also inSzabo et al., (1995) Curr Opin Struct Biol 5, 699-705, the disclosuresof which are incorporated by reference. This technique permits thedetection of interactions between molecules in real time, without theneed of labeled molecules. This technique is based on the surfaceplasmon resonance (SPR) phenomenon. Briefly, the candidate ligandmolecule to be tested is attached to a surface (such as a carboxymethyldextran matrix). A light beam is directed towards the side of thesurface that does not contain the sample to be tested and is reflectedby said surface. The SPR phenomenon causes a decrease in the intensityof the reflected light with a specific association of angle andwavelength. The binding of candidate ligand molecules cause a change inthe refraction index on the surface, which change is detected as achange in the SPR signal. For screening of candidate ligand molecules orsubstances that are able to interact with the GENSET protein, or afragment thereof, the GENSET protein, or a fragment thereof, isimmobilized onto a surface. This surface comprises one side of a cellthrough which flows the candidate molecule to be assayed. The binding ofthe candidate molecule on the GENSET protein, or a fragment thereof, isdetected as a change of the SPR signal. The candidate molecules testedmay be proteins, peptides, carbohydrates, lipids, or small moleculesgenerated by combinatorial chemistry. This technique may also beperformed by immobilizing eukaryotic or prokaryotic cells or lipidvesicles exhibiting an endogenous or a recombinantly expressed GENSETprotein at their surface.

The main advantage of the method is that it allows the determination ofthe association rate between the GENSET protein and moleculesinteracting with the GENSET protein. It is thus possible to selectspecifically ligand molecules interacting with the GENSET protein, or afragment thereof, through strong or conversely weak associationconstants.

E. Candidate Ligands Obtained through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), which disclosure is herebyincorporated by reference in its entirety, and relies upon the fusion ofa bait protein to the DNA binding domain of the yeast Gal4 protein. Thistechnique is also described in the U.S. Pat. No. 5,667,973 and the U.S.Pat. No. 5,283,173, the technical teachings of both patents being hereinincorporated by reference.

The general procedure of library screening by the two-hybrid assay maybe performed as described by Harper et al., (1993), Cell, 75: 805-816 oras described by Cho et al., (1998), Proc. Natl. Acad. Sci. USA,95(7):3752-3757 or also Fromont-Racine et al., (1997), Nature Genetics,16(3): 277-282, which disclosures are hereby incorporated by referencein their entireties.

The bait protein or polypeptide comprises a polypeptide of the presentinvention.

More precisely, the nucleotide sequence encoding the GENSET polypeptideor a fragment or variant thereof is fused to a polynucleotide encodingthe DNA binding domain of the GAL4 protein, the fused nucleotidesequence being inserted in a suitable expression vector, for examplepAS2 or pM3.

Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein. Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “prey” polypeptides.

A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but nonlimiting example the two different yeast strains may be the followings:

-   190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901,    his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3,    cyh^(r));-   187, the phenotype of which is (MATa gal4 gal80 his3 trp1-901    ade2-101 ura3-52 leu2-3,-   112 URA3 GAL-lacZmet⁻), which is the opposite mating type of Y190.

Briefly, 20 μg of pAS2/GENSET and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine synthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, beta-gal⁺) are then grown on plateslacking histidine, leucine, but containing tryptophan and cycloheximide(10 mg/ml) to select for loss of pAS2/GENSET plasmids but retention ofpACT-cDNA library plasmids. The resulting Y190 strains are mated withY187 strains expressing GENSET or non-related control proteins; such ascyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper etal. (1993) and by Brain et al., (1993), Mol. Cell Biol., 13:4760-4769,which disclosures are hereby incorporated by reference in theirentireties, and screened for beta galactosidase by filter lift assay.Yeast clones that are beta gal-after mating with the control Gal4fusions are considered false positives.

In another embodiment of the two-hybrid method according to theinvention, interaction between the GENSET or a fragment or variantthereof with cellular proteins may be assessed using the Matchmaker TwoHybrid System 2 (Catalog No. K1604-1, Clontech). As described in themanual accompanying the kit, the disclosure of which is incorporatedherein by reference, nucleic acids encoding the GENSET protein or aportion thereof, are inserted into an expression vector such that theyare in frame with DNA encoding the DNA binding domain of the yeasttranscriptional activator GAL4. A desired cDNA, preferably human cDNA,is inserted into a second expression vector such that they are in framewith DNA encoding the activation domain of GAL4. The two expressionplasmids are transformed into yeast and the yeast are plated onselection medium which selects for expression of selectable markers oneach of the expression vectors as well as GAL4 dependent expression ofthe HIS3 gene. Transformants capable of growing on medium lackinghistidine are screened for GAL4 dependent lacZ expression. Those cellswhich are positive in both the histidine selection and the lacZ assaycontain interaction between GENSET and the protein or peptide encoded bythe initially selected cDNA insert.

Compounds Modulating GENSET Biological Activity

Another method of screening for compounds that modulate GENSETexpression and/or biological activity is by measuring the effects oftest compounds on specific biological activity, e.g. a GENSET biologicalactivity in a host cell. In one embodiment, the present inventionrelates to a method of identifying an agent which alters GENSETbiological activity, wherein a nucleic acid construct comprising anucleic acid which encodes a mammalian GENSET polypeptide is introducedinto a host cell. The host cells produced are maintained underconditions appropriate for expression of the encoded mammalian GENSETpolypeptides, whereby the nucleic acid is expressed. The host cells arethen contacted with a compound to be assessed (an “agent,” or “testagent”), and the properties of the cells are assessed. Detection of achange in any GENSET polypeptide-associated property in the presence ofthe agent indicates that the agent alters GENSET activity. In aparticular embodiment, the invention relates to a method of identifyingan agent which is an activator of GENSET activity, wherein detection ofan increase of any GENSET polypeptide-associated property in thepresence of the agent indicates that the agent activates GENSETactivity. In another particular embodiment, the invention relates to amethod of identifying an agent which is an inhibitor of GENSET activity,wherein detection of a decrease of any GENSET polypeptide-associatedproperty in the presence of the agent indicates that the agent inhibitsGENSET activity.

In a particular embodiment, a high throughput screen can be used toidentify agents that activate (enhance) or inhibit GENSET activity (Seee.g., PCT publication WO 98/45438, which disclosure is herebyincorporated by reference in its entirety). For example, the method ofidentifying an agent which alters GENSET activity can be performed asfollows. A nucleic acid construct comprising a polynucleotide whichencodes a mammalian GENSET polypeptide is introduced into a host cell toproduce recombinant host cells. The recombinant host cells are thenmaintained under conditions appropriate for expression of the encodedmammalian GENSET polypeptide, whereby the nucleic acid is expressed. Thecompound to be assessed is added to the recombinant host cells; theresulting combination is referred to as a test sample. A detectable,GENSET polypeptide-associated property of the cells is detected. Acontrol can be used in the methods of detecting agents which alterGENSET activity. For example, the control sample includes the samereagents but lacks the compound or agent being assessed; it is treatedin the same manner as the test sample.

Methods of Screening for Compounds Modulating GENSET Expression and/orActivity

The present invention also relates to methods of screening compounds fortheir ability to modulate (e.g. increase or inhibit) the activity orexpression of GENSET. More specifically, the present invention relatesto methods of testing compounds for their ability either to increase orto decrease expression or activity of GENSET. The assays are performedin vitro or in vivo.

In Vitro Methods

In vitro, cells expressing GENSET polypeptides are incubated in thepresence and absence of the test compound. By determining the level ofGENSET expression in the presence of the test compound or the level ofGENSET activity in the presence of the test compound, compounds can beidentified that suppress or enhance GENSET expression or activity.Alternatively, constructs comprising a GENSET regulatory sequenceoperably linked to a reporter gene (e.g. luciferase, chloramphenicolacetyl transferase, LacZ, green fluorescent protein, etc.) can beintroduced into host cells and the effect of the test compounds onexpression of the reporter gene detected. Cells suitable for use in theforegoing assays include, but are not limited to, cells having the sameorigin as tissues or cell lines in which the polypeptide has beendetermined to be expressed by methods common to the art such asdiscussed herein. Consequently, the present invention encompasses amethod for screening molecules that modulate the expression of a GENSETgene, said screening method comprising the steps of:

-   -   a) cultivating a prokaryotic or an eukaryotic cell that has been        transfected with a nucleotide sequence encoding a GENSET protein        or a variant or a fragment thereof, placed under the control of        its own promoter;    -   b) bringing into contact said cultivated cell with a molecule to        be tested;    -   c) quantifying the expression of said GENSET protein or a        variant or a fragment thereof in the presence of said molecule.

Using DNA recombination techniques well known by the one skill in theart, the GENSET protein encoding DNA sequence is inserted into anexpression vector, downstream from its promoter sequence. As anillustrative example, the promoter sequence of the GENSET gene iscontained in the 5′ untranscribed region of the GENSET genomic DNA.

The quantification of the expression of a GENSET protein may be realizedeither at the mRNA level (using for example Northen blots, RT-PCR,preferably quantitative RT-PCR with primers and probes specific for theGENSET mRNA of interest) or at the protein level (using polyclonal ormonoclonal antibodies in immunoassays such as ELISA or RIA assays,Western blots, or immunochemistry).

The present invention also concerns a method for screening substances ormolecules that are able to increase, or in contrast to decrease, thelevel of expression of a GENSET gene. Such a method may allow the oneskilled in the art to select substances exerting a regulating effect onthe expression level of a GENSET gene and which may be useful as activeingredients included in pharmaceutical compositions for treatingpatients suffering from disorders associated with abnormal levels ofGENSET products.

Thus, another part of the present invention is a method for screening acandidate molecule that modulates the expression of a GENSET gene, thismethod comprises the following steps:

-   -   a) providing a recombinant cell host containing a nucleic acid,        wherein said nucleic acid comprises a GENSET 5′ regulatory        region or a regulatory active fragment or variant thereof,        operably linked to a polynucleotide encoding a detectable        protein;    -   b) obtaining a candidate molecule; and    -   c) determining the ability of said candidate molecule to        modulate the expression levels of said polynucleotide encoding        the detectable protein.

In a further embodiment, said nucleic acid comprising a GENSET 5′regulatory region or a regulatory active fragment or variant thereof,includes the 5′ UTR region of a GENSET cDNA selected from the groupcomprising of the 5′ UTRs of the polynucleotide sequences of theSequence Listing, those of human cDNA clone inserts of the depositedclone pool, regulatory active fragments, and variants thereof. In a morepreferred embodiment of the above screening method, said nucleic acidincludes a promoter sequence which is endogenous with respect to theGENSET 5′ UTR sequence. In another more preferred embodiment of theabove screening method, said nucleic acid includes a promoter sequencewhich is exogenous with respect to the GENSET 5′ UTR sequence definedtherein.

Preferred polynucleotides encoding a detectable protein arepolynucleotides encoding beta galactosidase, green fluorescent protein(GFP) and chloramphenicol acetyl transferase (CAT).

The invention further relates to a method for the production of apharmaceutical composition comprising a method of screening a candidatemolecule that modulates the expression of a GENSET gene and furthermoremixing the identified molecule with a physiologically acceptablecarrier.

The invention also pertains to kits for the screening of a candidatesubstance modulating the expression of a GENSET gene. Preferably, suchkits comprise a recombinant vector that allows the expression of aGENSET 5′ regulatory region or a regulatory active fragment or a variantthereof, operably linked to a polynucleotide encoding a detectableprotein or a GENSET protein or a fragment or a variant thereof. Morepreferably, such kits include a recombinant vector that comprises anucleic acid including the 5′ UTR region of a GENSET cDNA selected fromthe group comprising the 5′ UTRs of the polynucleotide sequences of theSequence Listing, those of human cDNA clone inserts of the depositedclone pool, regulatory active fragments and variants thereof, beingoperably linked to a polynucleotide encoding a detectable protein.

For the design of suitable recombinant vectors useful for performing thescreening methods described above, it will be referred to the section ofthe present specification wherein the preferred recombinant vectors ofthe invention are detailed.

Another object of the present invention comprises methods and kits forthe screening of candidate substances that interact with a GENSETpolypeptide, fragments or variants thereof. By their capacity to bindcovalently or non-covalently to a GENSET protein, fragments or variantsthereof, these substances or molecules may be advantageously used bothin vitro and in vivo.

In vitro, said interacting molecules may be used as detection means inorder to identify the presence of a GENSET protein in a sample,preferably a biological sample.

A method for the screening of a candidate substance that interact with aGENSET polypeptide, fragments or variants thereof, said methodscomprising the following steps:

-   -   a) providing a polypeptide comprising, consisting essentially        of, or consisting of a GENSET protein or a fragment comprising a        contiguous span of at least 6 amino acids, preferably at least 8        to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,        40, 50, or 100 amino acids of a polypeptide of the present        invention;    -   b) obtaining a candidate substance;    -   c) bringing into contact said polypeptide with said candidate        substance;    -   d) detecting the complexes formed between said polypeptide and        said candidate substance.

The invention further relates to a method for the production of apharmaceutical composition comprising a method for the screening of acandidate substance that interact with a GENSET polypeptide, fragmentsor variants thereof and furthermore mixing the identified substance witha physiologically acceptable carrier.

The invention further concerns a kit for the screening of a candidatesubstance interacting with the GENSET polypeptide, wherein said kitcomprises:

-   -   a) polypeptide comprising, consisting essentially of, or        consisting of a GENSET protein or a fragment comprising a        contiguous span of at least 6 amino acids, preferably at least 8        to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,        40, 50, or 100 amino acids of a polypeptide of the present        invention; and    -   b) optionally means useful to detect the complex formed between        said polypeptide or a variant thereof and the candidate        substance.

In a preferred embodiment of the kit described above, the detectionmeans comprises a monoclonal or polyclonal antibody binding to saidGENSET protein or fragment or variant thereof.

In Vivo Methods

Compounds that suppress or enhance GENSET expression can also beidentified using in vivo screens. In these assays, the test compound isadministered (e.g. intravenously, intraperitoneally, intramuscularly,orally, or otherwise), to the animal, for example, at a variety of doselevels. The effect of the compound on GENSET expression is determined bycomparing GENSET levels, for example in tissues known to express thegene of interest using Northern blots, immunoassays, PCR, etc., asdescribed above. Suitable test animals include, but are not limited to,rodents (e.g., mice and rats), primates, and rabbits. Humanized mice canalso be used as test animals, that is mice in which the endogenous mouseprotein is ablated (knocked out) and the homologous human protein addedback by standard transgenic approaches. Such mice express only the humanform of a protein. Humanized mice expressing only the human GENSET canbe used to study in vivo responses to potential agents regulating GENSETprotein or mRNA levels. As an example, transgenic mice have beenproduced carrying the human apoE4 gene. They are then bred with a mouseline that lacks endogenous apoE, to produce an animal model carryinghuman proteins believed to be instrumental in development of Alzheimer'spathology. Such transgenic animals are useful for dissecting thebiochemical and physiological steps of disease, and for development oftherapies for disease intervention (Loring, et al, 1996) (incorporatedherein by reference in its entirety).

Uses for Compounds Modulating GENSET Expression and/or BiologicalActivity

Using in vivo (or in vitro) systems, it may be possible to identifycompounds that exert a tissue specific effect, for example, thatincrease GENSET expression or activity only in tissues of interest, suchas the adrenal gland, bone marrow, brain, cerebellum, colon, fetalbrain, fetal kidney, fetal liver, heart, hypertrophic prostate, kidney,liver, lung, lymph ganglia, lymphocytes, muscle, ovary, pancreas,pituitary gland, placenta, prostate, salivary gland, spinal cord,spleen, stomach, intestine, substantia nigra, testis, thyroid, umbilicalcord, and uterus. Screening procedures such as those described above arealso useful for identifying agents for their potential use inpharmacological intervention strategies. Agents that enhance GENSET geneexpression or stimulate its activity may thus be used to induce anyphenotype associated with a GENSET gene, or to treat disorders resultingfrom a deficiency of a GENSET polypeptide activity or expression.Compounds that suppress GENSET polypeptide expression or inhibit itsactivity can be used to treat any disease or condition associated withincreased or deleterious GENSET polypeptide activity or expression.

Also encompassed by the present invention is an agent which interactswith a GENSET gene or polypeptide directly or indirectly, and inhibitsor enhances GENSET polypeptide expression and/or function. In oneembodiment, the agent is an inhibitor which interferes with a GENSETpolypeptide directly (e.g., by binding the GENSET polypeptide) orindirectly (e.g., by blocking the ability of the GENSET polypeptide tohave a GENSET biological activity). In a particular embodiment, aninhibitor of a GENSET protein is an antibody specific for the GENSETprotein or a functional portion of the GENSET protein; that is, theantibody binds a GENSET polypeptide. For example, the antibody can bespecific for a polypeptide encoded by one of the nucleic acid sequencesof human GENSET nucleic acids, a mammalian GENSET nucleic acid, orportions thereof. Alternatively, the inhibitor can be an agent otherthan an antibody (e.g., small organic molecule, protein or peptide)which binds the GENSET polypeptide and blocks its activity. For example,the inhibitor can be an agent which mimics the GENSET polypeptidestructurally, but lacks its function. Alternatively, it can be an agentwhich binds to or interacts with a molecule which the GENSET polypeptidenormally binds to or interacts with, thus blocking the GENSETpolypeptide from doing so and preventing it from exerting the effects itwould normally exert.

In another embodiment, the agent is an enhancer (activator) of a GENSETpolypeptide which increases the activity of the GENSET polypeptide(increases the effect of a given amount or level of GENSET), increasesthe length of time it is effective (by preventing its degradation orotherwise prolonging the time during which it is active) or both eitherdirectly or indirectly. For example, GENSET polynucleotides andpolypeptides can be used to identify drugs which increase or decreasethe ability of GENSET polypeptides to induce GENSET biological activity,which drugs are useful for the treatment or prevention of any disease orcondition associated with a GENSET biological activity.

The GENSET sequences of the present invention can also be used togenerate nonhuman gene knockout animals, such as mice, which lack aGENSET gene or transgenically overexpress a GENSET gene. For example,such GENSET gene knockout mice can be generated and used to obtainfurther insight into the function of the GENSET gene as well as assessthe specificity of GENSET activators and inhibitors. Also, overexpression of the GENSET gene (e.g., a human GENSET gene) in transgenicmice can be used as a means of creating a test system for GENSETactivators and inhibitors (e.g., against a human GENSET polypeptide). Inaddition, the GENSET gene can be used to clone the GENSETpromoter/enhancer in order to identify regulators of GENSET genetranscription. GENSET gene knockout animals include animals whichcompletely or partially lack the GENSET gene and/or GENSET activity orfunction. Thus the present invention relates to a method of inhibiting(partially or completely) a GENSET biological activity in a mammal(e.g., a human), the method comprising administering to the mammal aneffective amount of an inhibitor of a GENSET polypeptide orpolynucleotide. The invention also relates to a method of enhancing aGENSET biological activity in a mammal, the method comprisingadministering to the mammal an effective amount of an enhancer of aGENSET polypeptide or polynucleotide.

Inhibiting GENSET Gene Expression

Therapeutic compositions according to the present invention may compriseadvantageously one or several GENSET oligonucleotide fragments as anantisense tool or a triple helix tool that inhibits the expression ofthe corresponding GENSET gene.

Antisense Approach

In antisense approaches, nucleic acid sequences complementary to an mRNAare hybridized to the mRNA intracellularly, thereby blocking theexpression of the protein encoded by the mRNA. The antisense nucleicacid molecules to be used in gene therapy may be either DNA or RNAsequences. Preferred methods using antisense polynucleotide according tothe present invention are the procedures described by Sczakiel et al.,(1995) Trends Microbiol. 3(6):213-217, which disclosure is herebyincorporated by reference in its entirety.

Preferably, the antisense tools are chosen among the polynucleotides(15-200 bp long) that are complementary to GENSET mRNA, more preferablyto the 5′end of the GENSET mRNA. In another embodiment, a combination ofdifferent antisense polynucleotides complementary to different parts ofthe desired targeted gene are used.

Other preferred antisense polynucleotides according to the presentinvention are sequences complementary to either a sequence of GENSETmRNAs comprising the translation initiation codon ATG or a sequence ofGENSET genomic DNA containing a splicing donor or acceptor site.Preferably, the antisense polynucleotides of the invention have a 3′polyadenylation signal that has been replaced with a self-cleavingribozyme sequence, such that RNA polymerase II transcripts are producedwithout poly(A) at their 3′ ends, these antisense polynucleotides beingincapable of export from the nucleus, such as described by Liu et al.(1994), Proc. Natl. Acad. Sci. USA. 91: 4528-4262, which disclosure ishereby incorporated by reference in its entirety. In a preferredembodiment, these GENSET antisense polynucleotides also comprise, withinthe ribozyme cassette, a histone stem-loop structure to stabilizecleaved transcripts against 3′-5′ exonucleolytic degradation, such asthe structure described by Eckner et al., (1991) EMBO J. 10:3513-3522,which disclosure is hereby incorporated by reference in its entirety.

The antisense nucleic acids should have a length and melting temperaturesufficient to permit formation of an intracellular duplex havingsufficient stability to inhibit the expression of the GENSET mRNA in theduplex. Strategies for designing antisense nucleic acids suitable foruse in gene therapy are disclosed in Green et al., (1986) Ann. Rev.Biochem. 55:569-597 and Izant and Weintraub, (1984) Cell 36(4):1007-15,the disclosures of which are incorporated herein by reference.

In some strategies, antisense molecules are obtained by reversing theorientation of the GENSET coding region with respect to a promoter so asto transcribe the opposite strand from that which is normallytranscribed in the cell. The antisense molecules may be transcribedusing in vitro transcription systems such as those which employ T7 orSP6 polymerase to generate the transcript. Another approach involvestranscription of GENSET antisense nucleic acids in vivo by operablylinking DNA containing the antisense sequence to a promoter in asuitable expression vector. Alternatively, oligonucleotides which arecomplementary to the strand normally transcribed in the cell may besynthesized in vitro. Thus, the antisense nucleic acids arecomplementary to the corresponding mRNA and are capable of hybridizingto the mRNA to create a duplex.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH.sub.2 component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. Tne sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2. degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications (U.S. Pat. No.6,242,590, hereby incorporated by reference).

Various types of antisense oligonucleotides complementary to thesequence of the GENSET cDNA or genomic DNA may be used. In one preferredembodiment, stable and semi-stable antisense oligonucleotides describedin International Application No. PCT WO94/23026, hereby incorporated byreference, are used. In these molecules, the 3′ end or both the 3′ and5′ ends are engaged in intramolecular hydrogen bonding betweencomplementary base pairs. These molecules are better able to withstandexonuclease attacks and exhibit increased stability compared toconventional antisense oligonucleotides.

In another preferred embodiment, the antisense oligodeoxynucleotidesagainst herpes simplex virus types 1 and 2 described in InternationalApplication No. WO 95/0414, hereby incorporated by reference, are used.

In yet another preferred embodiment, the covalently cross-linkedantisense oligonucleotides described in International Application No. WO96/31523, hereby incorporated by reference, are used. These double- orsingle-stranded oligonucleotides comprise one or more, respectively,inter- or intra-oligonucleotide covalent cross-linkages, wherein thelinkage consists of an amide bond between a primary amine group of onestrand and a carboxyl group of the other strand or of the same strand,respectively, the primary amine group being directly substituted in the2′ position of the strand nucleotide monosaccharide ring, and thecarboxyl group being carried by an aliphatic spacer group substituted ona nucleotide or nucleotide analog of the other strand or the samestrand, respectively.

The antisense oligodeoxynucleotides and oligonucleotides disclosed inInternational Application No. WO 92/18522, incorporated by reference,may also be used. These molecules are stable to degradation and containat least one transcription control recognition sequence which binds tocontrol proteins and are effective as decoys therefor. These moleculesmay contain “hairpin” structures, “dumbbell” structures, “modifieddumbbell” structures, “cross-linked” decoy structures and “loop”structures.

In another preferred embodiment, the cyclic double-strandedoligonucleotides described in European Patent Application No. 0 572 287A2, hereby incorporated by reference are used. These ligatedoligonucleotide “dumbbells” contain the binding site for a transcriptionfactor and inhibit expression of the gene under control of thetranscription factor by sequestering the factor.

Use of the closed antisense oligonucleotides disclosed in InternationalApplication No. WO 92/19732, hereby incorporated by reference, is alsocontemplated. Because these molecules have no free ends, they are moreresistant to degradation by exonucleases than are conventionaloligonucleotides. These oligonucleotides may be multifunctional,interacting with several regions which are not adjacent to the targetmRNA.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA (1989) 86: 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let. (1994) 4:1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. (1992)660:306-309; Manoharan et al., Bioorg. Med. Chem. Let. (1993)3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.(1992) 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J. (1991) 10:1111-1118; Kabanovet al., FEBS Lett. (1990) 259: 327-330; Svinarchuk et al., Biochimie(1993) 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res. (1990) 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides (1995) 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.(1995) 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta (1995) 1264:229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther. (1996) 277:923-937; U.S. Pat. No. 6,242,590, whichdisclosures are hereby incorporated by reference in their entireties

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart (U.S. Pat. No. 6,242,590, hereby incorporated by reference).

Further included in the present invention is a method of high throughputscreening of antisense nucleic acids and modified versions thereof forbinding to targeted GENSET polynucleotide sequences or fragmentsthereof. This method is directed toward determining optimally targetedsequences and/or optimal species of targeting antisense molecules forbinding. A preferred method comprises the steps of: contacting a randompool of test molecules with a set array of GENSET polynucleotidesequences or fragments thereof; detecting and quantifying binding oftest molecules to said array; and purification and identification ofbinding test molecules as discussed in U.S. Pat. No. 6,022,691, whichdisclosure is hereby incorporated by reference. Preferred test moleculesare antisense oligonucleotides, oligonucleosides, and modified versionsthereof as discussed herein. Further preferred test molecules are thosethat are capable of forming hydrogen bonds with GENSET polynucleotidesequences or fragments thereof.

The appropriate level of antisense nucleic acids required to inhibitgene expression may be determined using in vitro expression analysis.The antisense molecule may be introduced into the cells by diffusion,injection, infection or transfection using procedures known in the art.For example, the antisense nucleic acids can be introduced into the bodyas a bare or naked oligonucleotide, oligonucleotide encapsulated inlipid, oligonucleotide sequence encapsidated by viral protein, or as anoligonucleotide operably linked to a promoter contained in an expressionvector. The expression vector may be any of a variety of expressionvectors known in the art, including retroviral or viral vectors, vectorscapable of extrachromosomal replication, or integrating vectors. Thevectors may be DNA or RNA.

The antisense compounds of the invention encompass any physiologicallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and physiologically acceptable salts of the compounds of theinvention, physiologically acceptable salts of such prodrugs, and otherbioequivalents as discussed herein.

The antisense molecules are introduced onto cell samples at a number ofdifferent concentrations preferably between 1×10⁻¹⁰M to 1×10⁻⁴M. Oncethe minimum concentration that can adequately control gene expression isidentified, the optimized dose is translated into a dosage suitable foruse in vivo. For example, an inhibiting concentration in culture of1×10⁻⁷M translates into a dose of approximately 0.6 mg/kg bodyweight.Levels of oligonucleotide approaching 100 mg/kg bodyweight or higher maybe possible after testing the toxicity of the oligonucleotide inlaboratory animals. It is additionally contemplated that cells from thevertebrate are removed, treated with the antisense oligonucleotide, andreintroduced into the vertebrate.

In a preferred application of this invention, the polypeptide encoded bythe gene is first identified, so that the effectiveness of antisenseinhibition on translation can be monitored using techniques that includebut are not limited to antibody-mediated tests such as RIAs and ELISA,functional assays, or radiolabeling.

An alternative to the antisense technology that is used according to thepresent invention comprises using ribozymes that will bind to a targetsequence via their complementary polynucleotide tail and that willcleave the corresponding RNA by hydrolyzing its target site (namely“hammerhead ribozymes”). Briefly, the simplified cycle of a hammerheadribozyme comprises (1) sequence specific binding to the target RNA viacomplementary antisense sequences; (2) site-specific hydrolysis of thecleavable motif of the target strand; and (3) release of cleavageproducts, which gives rise to another catalytic cycle. Indeed, the useof long-chain antisense polynucleotide (at least 30 bases long) orribozymes with long antisense arms are advantageous. A preferreddelivery system for antisense ribozyme is achieved by covalently linkingthese antisense ribozymes to lipophilic groups or to use liposomes as aconvenient vector. Preferred antisense ribozymes according to thepresent invention are prepared as described by Rossi et al., (1991)Pharmacol. Ther. 50:245-254 and Sczakiel et al. (1995), the specificpreparation procedures being referred to in said articles being hereinincorporated by reference.

Triple Helix Approach

The GENSET genomic DNA may also be used to inhibit the expression of theGENSET gene based on intracellular triple helix formation.

Triple helix oligonucleotides are used to inhibit transcription from agenome. They are particularly useful for studying alterations in cellactivity when it is associated with a particular gene. The GENSET cDNAsor genomic DNAs of the present invention or, more preferably, a fragmentof those sequences, can be used to inhibit gene expression inindividuals having diseases associated with expression of a particulargene. Similarly, a portion of the GENSET genomic DNA can be used tostudy the effect of inhibiting GENSET gene transcription within a cell.Traditionally, homopurine sequences were considered the most useful fortriple helix strategies. However, homopyrimidine sequences can alsoinhibit gene expression. Such homopyrimidine oligonucleotides bind tothe major groove at homopurine:homopyrimidine sequences. Thus, bothtypes of sequences from the GENSET genomic DNA are contemplated withinthe scope of this invention.

To carry out gene therapy strategies using the triple helix approach,the sequences of the GENSET genomic DNA are first scanned to identify10-mer to 20-mer homopyrimidine or homopurine stretches which could beused in triple-helix based strategies for inhibiting GENSET expression.Following identification of candidate homopyrimidine or homopurinestretches, their efficiency in inhibiting GENSET expression is assessedby introducing varying amounts of oligonucleotides containing thecandidate sequences into tissue culture cells which express the GENSETgene.

The oligonucleotides can be introduced into the cells using a variety ofmethods known to those skilled in the art, including but not limited tocalcium phosphate precipitation, DEAE-Dextran, electroporation,liposome-mediated transfection or native uptake.

Treated cells are monitored for altered cell function or reduced GENSETexpression using techniques such as Northern blotting, RNase protectionassays, or PCR based strategies to monitor the transcription levels ofthe GENSET gene in cells which have been treated with theoligonucleotide. The cell functions to be monitored are predicted basedupon the homologies of the target gene corresponding to the cDNA fromwhich the oligonucleotide was derived with known gene sequences thathave been associated with a particular function. The cell functions canalso be predicted based on the presence of abnormal physiology withincells derived from individuals with a particular inherited disease,particularly when the cDNA is associated with the disease usingtechniques described in the section entitled “Identification of genesassociated with hereditary diseases or drug response”.

The oligonucleotides which are effective in inhibiting gene expressionin tissue culture cells may then be introduced in vivo using thetechniques and at a dosage calculated based on the in vitro results, asdescribed in the section entitled “Antisense Approach”.

In some embodiments, the natural (beta) anomers of the oligonucleotideunits can be replaced with alpha anomers to render the oligonucleotidemore resistant to nucleases. Further, an intercalating agent such asethidium bromide, or the like, can be attached to the 3′ end of thealpha oligonucleotide to stabilize the triple helix. For information onthe generation of oligonucleotides suitable for triple helix formation.See Griffin et al., (1989) Science 245:967-971, which is herebyincorporated by this reference.

Treating GENSET Gene-Related Disorders

The present invention further relates to methods, uses of GENSETpolypeptides and polynucleotides, and uses of modulators of GENSETpolypeptides and polynucleotides, for treating diseases/disordersassociated with GENSET genes by increasing or decreasing GENSET geneactivity and/or expression. These methodologies can be effected usingcompounds selected using screening protocols such as those describedherein and/or by using the gene therapy and antisense approachesdescribed in the art and herein. Gene therapy can be used to effecttargeted expression of GENSET genes in any tissue, e.g. a tissueassociated with the disease or condition to be treated. The GENSETcoding sequence can be cloned into an appropriate expression vector andtargeted to a particular cell type(s) to achieve efficient, high levelexpression. Introduction of the GENSET coding sequence into target cellscan be achieved, for example, using particle mediated DNA delivery,[Haynes et al., (1996) J Biotechnol. 44(1-3):37-42 and Maurer et al.,(1999) Mol Membr Biol. 16(1):129-40], direct injection of naked DNA,[Levy et al., (1996) Gene Ther. 3(3):201-11; and Felgner (1996) Hum GeneTher. 7(15):1791-3], or viral vector mediate transport [Smith et al.,(1996) Antiviral Res. 32(2):99-115, Stone et al., (2000) J Endocrinol.164(2):103-18; Wu and Ataai (2000), Curr Opin Biotechnol. 11(2):205-8],each of which disclosures are hereby incorporated by reference in theirentireties. Tissue specific effects can be achieved, for example, in thecase of virus mediated transport by using viral vectors that are tissuespecific, or by the use of promoters that are tissue specific. Forinstance, any tissue-specific promoter may be used to achieve specificexpression, for example albumin promoters (liver specific; Pinkert etal., 1987 Genes Dev. 1:268-277), lymphoid specific promoters (Calame etal., 1988 Adv. Immunol. 43:235-275), promoters of T-cell receptors(Winoto et al., 1989 EMBO J. 8:729-733) and immunoglobulins (Banerji etal., 1983 Cell 33:729-740; Queen and Baltimore 1983 Cell 33:741-748),neuron-specific promoters (e.g. the neurofilament promoter; Byrne etal., 1989 Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specificpromoters (Edlunch et al., 1985 Science 230:912-916) or mammarygland-specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316and European Application Publication No. 264, 166).Developmentally-regulated promoters can also be used, such as the murinehomeobox promoters (Kessel et al., 1990 Science 249:374-379) or thealpha-fetoprotein promoter (Campes et al., 1989 Genes Dev. 3:537-546).

Combinatorial approaches can also be used to ensure that the GENSETcoding sequence is activated in the target tissue [Butt and Karathanasis(1995) Gene Expr. 4(6):319-36; Miller and Whelan, (1997) Hum Gene Ther.8(7):803-15], which disclosures are hereby incorporated by reference intheir entireties. Antisense oligonucleotides complementary to GENSETmRNA can be used to selectively diminish or ablate the expression of theprotein, for example, at sites of inflammation. More specifically,antisense constructs or antisense oligonucleotides can be used toinhibit the production of GENSET in high expressing cells such asdetermined by methods common to the art or discussed herein. AntisensemRNA can be produced by transfecting into target cells an expressionvector with the GENSET gene sequence, or a portion thereof, oriented inan antisense direction relative to the direction of transcription.Appropriate vectors include viral vectors, including retroviral,adenoviral, and adeno-associated viral vectors, as well as nonviralvectors. Tissue specific promoters can be used, as described supra.Alternatively, antisense oligonucleotides can be introduced directlyinto target cells to achieve the same goal. (See also other deliverymethodologies described herein in connection with gene therapy.).Oligonucleotides can be selected/designed to achieve a high level ofspecificity [Wagner, et al (1996), Nat Biotechnol. 14(7):840-4], whichdisclosure is hereby incorporated by reference in its entirety. Thetherapeutic methodologies described herein are applicable to both humanand non-human mammals (including cats and dogs).

Pharmaceutical and Physiologically Acceptable Compositions

The present invention also relates to pharmaceutical or physiologicallyacceptable compositions comprising, as active agent, the polypeptides,nucleic acids or antibodies of the invention. The invention also relatesto compositions comprising, as active agent, compounds selected usingthe above-described screening protocols. Such compositions include theactive agent in combination with a pharmaceutical or physiologicallyacceptable carriers such as a physiologically acceptable salt, ester, orsalt of such esters. In the case of naked DNA, the “carrier” may be goldparticles. The amount of active agent in the composition can vary withthe agent, the patient and the effect sought. Likewise, the dosingregimen can vary depending on the composition and the disease/disorderto be treated.

Therefore, the invention related to methods for the production ofpharmaceutical composition comprising a method for selecting an activeagent, compound, substance or molecule using any of the screening methoddescribed herein and furthermore mixing the identified active agent,compound, substance or molecule with a physiologically acceptablecarrier.

The term “physiologically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Physiologically acceptable base addition salts are formed with metals oramines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci. (1977) 66:1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a physiologically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable physiologically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Physiologically acceptable salts of compounds may alsobe prepared with a physiologically acceptable cation. Suitablephysiologically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.For oligonucleotides, preferred examples of physiologically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach ,et al.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to:parenteral, intracranial, intraorbital, intracapsular, intraspinal,intracisternal, intrapulmonary, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, or rectal means. In addition to the activeingredients, these pharmaceutical compositions may contain suitablephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack PublishingCo.Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing physiologically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as powders, tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers, such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitaniumdioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquidpolyethylene glycol with or without stabilizers.

Formulations suitable for pulmonary or respiratory delivery include drypowders, liquid solutions or suspensions suitable for nebulization, andpropellant formulations suitable for use in metered dose inhalers(MDI's). The preparation of such formulations is well described in thepatent, scientific, and medical literatures, and the followingdescriptions are intended to be exemplary only.

Dry powder formulations will have a particle size within a preferredrange for deposition within the alveolar region of the lung, typicallyfrom 0.5 .mu.m to 5 .mu.m. Respirable powders of pharmaceuticalcompositions within the preferred size range can be produced by avariety of conventional techniques, such as jet-milling, spray-drying,solvent precipitation, and the like. Dry powders can then beadministered to the patient in conventional dry powder inhalers (DPI's)that use the patient's inspiratory breath through the device to dispersethe powder or in air-assisted devices that use an external power sourceto disperse the powder into an aerosol cloud, as described in U.S. Pat.No. 5,458,135, the full disclosure of which is incorporated herein byreference.

Dry powder devices typically require a powder mass in the range fromabout 1 mg to 10 mg to produce a single aerosolized dose, which maynecessitate addition of a dry bulking powder to the pharmaceuticalformulation. Preferred dry bulking powders include sucrose, lactose,trehalose, human serum albumin (HSA), and glycine. Other suitable drybulking powders include cellobiose, dextrans, maltotriose, pectin,sodium citrate, sodium ascorbate, mannitol, and the like. Furthermore,stabilizing buffers and salts may be used. Other additives, such aschelating agents, peptidase inhibitors, and the like, which wouldfacilitate the biological activity of the pharmaceutical compositiononce it is dissolved within the lung would be appropriate. For example,ethylenediaminetetraacetic acid (ETDA) would be useful as a chelator fordivalent cations which are peptidase cofactors.

Liquid formulations for use in nebulizer systems preferably employslightly acidic buffers (pH 4-6) such as acetate, ascorbate, andcitrate, at concentrations of 5 mM to 50 mM. These buffers can act asantioxidants. Physiologically acceptable components to enhance ormaintain chemical stability include: antioxidants, chelating agents,protease inhibitors, isotonic modifiers, inert gases, and the like. Apreferred type of nebulizer suitable for delivering such liquidformulations is described in U.S. Pat. No. 5,458,135, the disclosure ofwhich is incorporated herein by reference.

For use in MDI's, the pharmaceutical composition will be dissolved orsuspended in a suitable aerosol propellant, such as a chlorofluorocarbon(CFC) or a hydrofluorocarbon (HFC). Suitable CFC's includetrichloromonofluoromethane (propellant 11), dichlorotetrafluoromethane(propellant 114), and dichlorodifluoromethane (propellant 12). SuitableHFC's include tetrafluoroethane (HFC-134a) and heptafluoropropane(HFC-227).

Preferably, for incorporation into the aerosol propellant, thepharmaceutical composition will be processed into respirable particlesas described for the dry powder formulations. The particles are thensuspended in the propellant, typically being coated with a surfactant toenhance their dispersion. Suitable surfactants include oleic acid,sorbitan trioleate, and various long chain diglycerides andphospholipids. Such aerosol propellant formulations may further includea lower alcohol, such as ethanol (up to 30% by weight) and otheradditives to maintain or enhance chemical stability and physiologicalacceptability (U.S. Pat. No. 6,080,721, which disclosure is herebyincorporated by reference in its entirety).

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethylcellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of a GENSET polypeptide, such labelingwould include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example a GENSET polypeptide or fragments thereof,antibodies specific to GENSET polypeptides, agonists, antagonists orinhibitors of GENSET polypeptides, which ameliorates the symptoms orcondition. Therapeutic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Other factors that maybe considered when evaluating the proper dosage include the chemicalnature of the compound destined for delivery, the biological responsesassociated with the compound (both intended and coincidental) andanticipated contraindications. Additionally, the mode of delivery(including but not limited to systemic and/or local applications: oral,oral enteric, intramuscular injection, subcutaneous injection,intradermal injection, interarticular space, intravascular injection,intravenous infusion, suppository, topical preparation, transdermalsystem), the duration and frequency of administration (e.g. n doses perhours, n doses per day, n doses per week, cumulative dosage per day,cumulative dosage per week), the biologically effective dose deliveredto target site, often indicated by plasma level concentrations, and therate or efficiency of compound clearance from the body may beconsidered. Long-acting pharmaceutical compositions maybe administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc. In general, for a 75 kgindividual the normal dosage range are as follows: for a small moleculecompound an effective does is usually between 0.3-50 mg/kg; forrecombinant polypeptides an effective dose is usually between 0.25-7.5mg/kg; for compounds used for mediating humoral immune responses (e.g.,polyvalent pneumococcal vaccine, Rh₀ (D) immune globulin, Hepatitis Bvaccine, anti-CD20 antigen) the effective dose is usually between0.0015-1.5 mg/kg; for hormone supplemental compounds (e.g. estradiol,norethindrone) the effective dose is usually between 0.0005-0.5 mg/kgdepending upon delivery system utilized (e.g. transdermal, oral,topical).

Transdermal delivery systems (e.g. estradiol transdermal system,transdermal scopolamine system, transfermal nicotine patch) must becalibrated for nominal delivery dosages based upon efficiency ofpercutaneous delivery for the individual and specific compounds, surfacearea (cm²) of transdermal system contact, quantity and form of compoundintegrated into transdermal delivery system and anatomical location ofpositioned transdermal system. The effective dosage range of compoundsadmistered in this manner is usually between 0.005-0.5 mg/kg

Uses of Genset Sequences: Computer-Related Embodiments

As used herein the term “GENSET cDNAs” encompasses the nucleotidesequences of the present invention.

It will be appreciated by those skilled in the art that the nucleic acidcodes of the invention and polypeptide codes of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any of the presently knownmethods for recording information on a computer readable medium togenerate manufactures comprising one or more of the nucleic acid codesof the invention, or one or more of the polypeptide codes of theinvention. Another aspect of the present invention is a computerreadable medium having recorded thereon at least 1, 2, 5, 10, 15, 20,25, 30, or 50 nucleic acid codes of the invention. Another aspect of thepresent invention is a computer readable medium having recorded thereonat least 1, 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of theinvention.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularlycomputer systems which store and manipulate the sequence informationdescribed herein. One example of a computer system 100 is illustrated inblock diagram form in FIG. 1. As used herein, “a computer system” refersto the hardware components, software components, and data storagecomponents used to analyze the nucleotide sequences of the nucleic acidcodes of the invention or the amino acid sequences of the polypeptidecodes of the invention. In one embodiment, the computer system 100 is aSun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). Thecomputer system 100 preferably includes a processor for processing,accessing and manipulating the sequence data. The processor 105 can beany well-known type of central processing unit, such as the Pentium IIIfrom Intel Corporation, or similar processor from Sun, Motorola, Compaqor International Business Machines.

Preferably, the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data, and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular embodiment, the computer system 100 includes aprocessor 105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some embodiments, the computer system100 further includes one or more data retrieving device 118 for readingthe data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, etc. In someembodiments, the internal data storage device 110 is a removablecomputer readable medium such as a floppy disk, a compact disk, amagnetic tape, etc. containing control logic and/or data recordedthereon. The computer system 100 may advantageously include or beprogrammed by appropriate software for reading the control logic and/orthe data from the data storage component once inserted in the dataretrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of thenucleic acid codes of the invention or the amino acid sequences of thepolypeptide codes of the invention (such as search tools, compare tools,and modeling tools etc.) may reside in main memory 115 during execution.

In some embodiments, the computer system 100 may further comprise asequence comparer for comparing the above-described nucleic acid codesof the invention or the polypeptide codes of the invention stored on acomputer readable medium to reference nucleotide or polypeptidesequences stored on a computer readable medium. A “sequence comparer”refers to one or more programs which are implemented on the computersystem 100 to compare a nucleotide or polypeptide sequence with othernucleotide or polypeptide sequences and/or compounds including but notlimited to peptides, peptidomimetics, and chemicals stored within thedata storage means. For example, the sequence comparer may compare thenucleotide sequences of nucleic acid codes of the invention or the aminoacid sequences of the polypeptide codes of the invention stored on acomputer readable medium to reference sequences stored on a computerreadable medium to identify homologies, motifs implicated in biologicalfunction, or structural motifs. The various sequence comparer programsidentified elsewhere in this patent specification are particularlycontemplated for use in this aspect of the invention.

FIG. 2 is a flow diagram illustrating one embodiment of a process 200for comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK, PIR OR SWISSPROT that is availablethrough the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the present invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid code of the invention or a polypeptide code of theinvention, a data storage device having retrievably stored thereonreference nucleotide sequences or polypeptide sequences to be comparedto the nucleic acid code of the invention or polypeptide code of theinvention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify motifs implicated in biological function andstructural motifs in the nucleic acid code of the invention andpolypeptide codes of the invention or it may identify structural motifsin sequences which are compared to these nucleic acid codes andpolypeptide codes. In some embodiments, the data storage device may havestored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50of the nucleic acid codes of the invention or polypeptide codes of theinvention.

Another aspect of the present invention is a method for determining thelevel of homology between a nucleic acid code of the invention and areference nucleotide sequence, comprising the steps of reading thenucleic acid code and the reference nucleotide sequence through the useof a computer program which determines homology levels and determininghomology between the nucleic acid code and the reference nucleotidesequence with the computer program. The computer program may be any of anumber of computer programs for determining homology levels, includingthose specifically enumerated herein, including BLAST2N with the defaultparameters or with any modified parameters. The method may beimplemented using the computer systems described above. The method mayalso be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of theabove described nucleic acid codes of the invention through the use ofthe computer program and determining homology between the nucleic acidcodes and reference nucleotide sequences.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 ina computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it should be in the single letter amino acid codeso that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are no more characters to read, then the process 250 moves to astate 276 wherein the level of homology between the first and secondsequences is displayed to the user. The level of homology is determinedby calculating the proportion of characters between the sequences thatwere the same out of the total number of sequences in the firstsequence. Thus, if every character in a first 100 nucleotide sequencealigned with a every character in a second sequence, the homology levelwould be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of the nucleic acid codes of thepresent invention, to reference nucleotide sequences in order todetermine whether the nucleic acid code of the invention differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or the nucleic acid code of the invention. Inone embodiment, the computer program may be a program which determineswhether the nucleotide sequences of the nucleic acid codes of theinvention contain one or more single nucleotide polymorphisms (SNP) withrespect to a reference nucleotide sequence. These single nucleotidepolymorphisms may each comprise a single base substitution, insertion,or deletion.

Another aspect of the present invention is a method for determining thelevel of homology between a polypeptide code of the invention and areference polypeptide sequence, comprising the steps of reading thepolypeptide code of the invention and the reference polypeptide sequencethrough use of a computer program which determines homology levels anddetermining homology between the polypeptide code and the referencepolypeptide sequence using the computer program.

Accordingly, another aspect of the present invention is a method fordetermining whether a nucleic acid code of the invention differs at oneor more nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of theinvention and the reference nucleotide sequences through the use of thecomputer program and identifying differences between the nucleic acidcodes and the reference nucleotide sequences with the computer program.

In other embodiments the computer based system may further comprise anidentifier for identifying features within the nucleotide sequences ofthe nucleic acid codes of the invention or the amino acid sequences ofthe polypeptide codes of the invention. An “identifier” refers to one ormore programs which identifies certain features within theabove-described nucleotide sequences of the nucleic acid codes of theinvention or the amino acid sequences of the polypeptide codes of theinvention. In one embodiment, the identifier may comprise a programwhich identifies an open reading frame in the cDNAs codes of theinvention.

FIG. 4 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group (www.gcg.com).

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence.

It should be noted, that if the feature attribute is not found in thefirst sequence at the decision state 316, the process 300 moves directlyto the decision state 320 in order to determine if any more featuresexist in the database.

In another embodiment, the identifier may comprise a molecular modelingprogram which determines the 3-dimensional structure of the polypeptidescodes of the invention. Such programs may use any methods known to thoseskilled in the art including methods based on homology-modeling, foldrecognition and ab initio methods as described in Sternberg et al,(1999) Curr Opin Struct Biol. 9(3):368-73, which disclosure is herebyincorporated by reference in its entirety. In some embodiments, themolecular modeling program identifies target sequences that are mostcompatible with profiles representing the structural environments of theresidues in known three-dimensional protein structures. (See, e.g.,Eisenberg et al., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, whichdisclosure is hereby incorporated by reference in its entirety). Inanother technique, the known three-dimensional structures of proteins ina given family are superimposed to define the structurally conservedregions in that family. This protein modeling technique also uses theknown three-dimensional structure of a homologous protein to approximatethe structure of the polypeptide codes of the invention. (See e.g.,Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, whichdisclosure is hereby incorporated by reference in its entirety).Conventional homology modeling techniques have been used routinely tobuild models of proteases and antibodies. [Sowdhamini et al. (1997),Protein Engineering 10:207, 215]. Comparative approaches can also beused to develop three-dimensional protein models when the protein ofinterest has poor sequence identity to template proteins. In some cases,proteins fold into similar three-dimensional structures despite havingvery weak sequence identities. For example, the three-dimensionalstructures of a number of helical cytokines fold in similarthree-dimensional topology in spite of weak sequence homology.

The recent development of threading methods now enables theidentification of likely folding patterns in a number of situationswhere the structural relatedness between target and template(s) is notdetectable at the sequence level. Hybrid methods, in which foldrecognition is performed using Multiple Sequence Threading (MST),structural equivalencies are deduced from the threading output using adistance geometry program DRAGON to construct a low resolution model,and a full-atom representation is constructed using a molecular modelingpackage such as QUANTA.

According to this 3-step approach, candidate templates are firstidentified by using the novel fold recognition algorithm MST, which iscapable of performing simultaneous threading of multiple alignedsequences onto one or more 3-D structures. In a second step, thestructural equivalencies obtained from the MST output are converted intointerresidue distance restraints and fed into the distance geometryprogram DRAGON, together with auxiliary information obtained fromsecondary structure predictions. The program combines the restraints inan unbiased manner and rapidly generates a large number of lowresolution model confirmations. In a third step, these low resolutionmodel confirmations are converted into full-atom models and subjected toenergy minimization using the molecular modeling package QUANTA. (Seee.g., Aszódi et al., (1997) Proteins: Structure, Function, and Genetics,Supplement 1:38-42).

The results of the molecular modeling analysis may then be used inrational drug design techniques to identify agents which modulate theactivity of the polypeptide codes of the invention.

Accordingly, another aspect of the present invention is a method ofidentifying a feature within the nucleic acid codes of the invention orthe polypeptide codes of the invention comprising reading the nucleicacid code(s) or the polypeptide code(s) through the use of a computerprogram which identifies features therein and identifying featureswithin the nucleic acid code(s) or polypeptide code(s) with the computerprogram. In one embodiment, computer program comprises a computerprogram which identifies open reading frames. In a further embodiment,the computer program identifies linear or structural motifs in apolypeptide sequence. In another embodiment, the computer programcomprises a molecular modeling program. The method may be performed byreading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 ofthe nucleic acid codes of the invention or the polypeptide codes of theinvention through the use of the computer program and identifyingfeatures within the nucleic acid codes or polypeptide codes with thecomputer program.

The nucleic acid codes of the invention or the polypeptide codes of theinvention may be stored and manipulated in a variety of data processorprograms in a variety of formats. For example, they may be stored astext in a word processing file, such as Microsoft WORD or WORDPERFECT oras an ASCII file in a variety of database programs familiar to those ofskill in the art, such as DB2, SYBASE, or ORACLE. In addition, manycomputer programs and databases may be used as sequence comparers,identifiers, or sources of reference nucleotide or polypeptide sequencesto be compared to the nucleic acid codes of the invention or thepolypeptide codes of the invention. The following list is intended notto limit the invention but to provide guidance to programs and databaseswhich are useful with the nucleic acid codes of the invention or thepolypeptide codes of the invention. The programs and databases which maybe used include, but are not limited to: MacPattern (EMBL),DiscoveryBase (Molecular Applications Group), GeneMine (MolecularApplications Group), Look (Molecular Applications Group), MacLook(Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN andBLASTX (Altschul et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB(Brutlag et al., 1990), Catalyst (Molecular Simulations Inc.),Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (MolecularSimulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II,(Molecular Simulations Inc.), Discover (Molecular Simulations Inc.),CHARMM (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the EMBL/Swissprotein database, the MDL Available ChemicalsDirectory database, the MDL Drug Data Report data base, theComprehensive Medicinal Chemistry database, Derwents's World Drug Indexdatabase, the BioByteMasterFile database, the Genbank database, and theGenseqn database. Many other programs and data bases would be apparentto one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Conclusion

As discussed above, the GENSET polynucleotides and polypeptides of thepresent invention or fragments thereof can be used for various purposes.The polynucleotides can be used to express recombinant protein foranalysis, characterization or therapeutic use; as markers for tissues inwhich the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in disease states); as molecular weight markers onSouthern gels; as chromosome markers or tags (when labeled) to identifychromosomes or to map related gene positions; as a reagent (including alabeled reagent) in assays designed to quantitatively determine levelsof GENSET expression in biological samples; to compare with endogenousDNA sequences in patients to identify potential genetic disorders; asprobes to hybridize and thus discover novel, related DNA sequences; as asource of information to derive PCR primers for genetic fingerprinting;for selecting and making oligomers for attachment to a “gene chip” orother support, including for examination for expression patterns; toraise anti-protein antibodies using DNA immunization techniques; and asan antigen to raise anti-DNA antibodies or elicit another immuneresponse. Where the polynucleotide encodes a protein which binds orpotentially binds to another protein (such as, for example, in areceptor-ligand interaction), the polynucleotide can also be used ininteraction trap assays (such as, for example, that described in Gyuriset al., (1993) Cell 75:791-803 to identify polynucleotides encoding theother protein with which binding occurs or to identify inhibitors of thebinding interaction.

The proteins or polypeptides provided by the present invention cansimilarly be used in assays to determine biological activity, includingin a panel of multiple proteins for high-throughput screening; to raiseantibodies or to elicit another immune response; as a reagent (includingthe labeled reagent) in assays designed to quantitatively determinelevels of the protein (or its receptor) in biological fluids; as markersfor tissues in which the corresponding protein is preferentiallyexpressed (either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); and, of course,to isolate correlative receptors or ligands. Where the protein binds orpotentially binds to another protein (such as, for example, in areceptor-ligand interaction), the protein can be used to identify theother protein with which binding occurs or to identify inhibitors of thebinding interaction. Proteins involved in these binding interactions canalso be used to screen for peptide or small molecule inhibitors oragonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning; A Laboratory Manual”, 2d ed., Cole SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology; Guide to Molecular CloningTechniques”, Academic Press, Berger and Kimmel eds., 1987, whichdisclosures are hereby incorporated by reference in their entireties.

Although this invention has been described in terms of certain preferredembodiments, other embodiments which will be apparent to those ofordinary skill in the art in view of the disclosure herein are alsowithin the scope of this invention. Accordingly, the scope of theinvention is intended to be defined only by reference to the appendedclaims.

EXAMPLES Example 1

Preparation of Antibody Compositions to the GENSET Protein

Substantially pure protein or polypeptide is isolated from transfectedor transformed cells containing an expression vector encoding the GENSETprotein or a portion thereof. The concentration of protein in the finalpreparation is adjusted, for example, by concentration on an Amiconfilter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes in the GENSET protein or a portionthereof can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein, (1975) Nature 256:495 orderivative methods thereof. Also see Harlow and Lane. (1988).

Briefly, a mouse is repetitively inoculated with a few micrograms of theGENSET protein, or a portion thereof, over a period of a few weeks. Themouse is then sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, (1980) Meth. Enzymol. 70:419, which disclosure is herebyincorporated by reference in its entirety, and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Davis, et al. (1986) Section 21-2.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes inthe GENSET protein or a portion thereof can be prepared by immunizingsuitable non-human animal with the GENSET protein or a portion thereof,which can be unmodified or modified to enhance immunogenicity. Asuitable non-human animal is preferably a non-human mammal is selected,usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crudepreparation which has been enriched for GENSET concentration can be usedto generate antibodies. Such proteins, fragments or preparations areintroduced into the non-human mammal in the presence of an appropriateadjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in theart. In addition the protein, fragment or preparation can be pretreatedwith an agent which will increase antigenicity, such agents are known inthe art and include, for example, methylated bovine serum albumin(mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, andkeyhole limpet hemocyanin (KLH). Serum from the immunized animal iscollected, treated and tested according to known procedures. If theserum contains polyclonal antibodies to undesired epitopes, thepolyclonal antibodies can be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factorsrelated both to the antigen and the host species. Also, host animalsvary in response to site of inoculations and dose, with both inadequateor excessive doses of antigen resulting in low titer antisera. Smalldoses (ng level) of antigen administered at multiple intradermal sitesappears to be most reliable. Techniques for producing and processingpolyclonal antisera are known in the art. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al., (1971) J. Clin.Endocrinol. Metab. 33:988-991, which disclosure is hereby incorporatedby reference in its entirety.

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al., (1973) Chap. 19 in: Handbook ofExperimental Immunology D. Wier (ed) Blackwell, which disclosure ishereby incorporated by reference in its entirety. Plateau concentrationof antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about12 uM). Affinity of the antisera for the antigen is determined bypreparing competitive binding curves, as described, for example, byFisher, (1980) Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Roseand Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C., whichdisclosure is hereby incorporated by reference in its entirety.

Antibody preparations prepared according to either the monoclonal or thepolyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

TABLE I SEQ ID Sequence ATCC ATCC NO. Type Clone ID_Clone Name NameDeposit Deposit Date 1 DNA 223583_114-044-2-0-E11-F S-100A10rP PTA-2732Nov. 27, 2000 2 Protein 223583_114-044-2-0-E11-F S-100A10rP PTA-2732Nov. 27, 2000 3 DNA 1000848582_181-40-4-0- SCPhx PTA-2732 Nov. 27, 2000A11-F 4 Protein 1000848582_181-40-4-0- SCPhx PTA-2732 Nov. 27, 2000A11-F 5 DNA 1000839315_220-26-1-0-F3- Chimerin PTA-2732 Nov. 27, 2000 F6 Protein 1000839315_220-26-1-0-F3- Chimerin PTA-2732 Nov. 27, 2000 F 7DNA 1000770704_208-27-3-0-G6- CalX PTA-2732 Nov. 27, 2000 F 8 Protein1000770704_208-27-3-0-G6- CalX PTA-2732 Nov. 27, 2000 F 9 DNA147103_106-024-1-0-H6-F sLRP10 PTA-2534 Sep. 27, 2000 10 Protein147103_106-024-1-0-H6-F sLRP10 PTA-2534 Sep. 27, 2000 11 DNA224168_116-096-3-0-G11-F sLRP10 PTA-2534 Sep. 27, 2000 12 Protein224168_116-096-3-0-G11-F sLRP10 PTA-2534 Sep. 27, 2000 13 DNA243303_116-118-4-0-A3-F sLRP10 PTA-2534 Sep. 27, 2000 14 Protein243303_116-118-4-0-A3-F sLRP10 PTA-2534 Sep. 27, 2000 15 DNA225432_116-083-3-0-C6-F sLRP10 PTA-2534 Sep. 27, 2000 16 Protein225432_116-083-3-0-C6-F sLRP10 PTA-2534 Sep. 27, 2000 17 DNA229633_114-049-1-0-D3-F STAMSAP PTA-2534 Sep. 27, 2000 18 Protein229633_114-049-1-0-D3-F STAMSAP PTA-2534 Sep. 27, 2000 19 DNA158523_106-030-2-0-A3-F OAR PTA-2732 Nov. 27, 2000 20 Protein158523_106-030-2-0-A3-F OAR PTA-2732 Nov. 27, 2000 21 DNA589198_184-11-1-0-E4-F COVI PTA-2732 Nov. 27, 2000 22 Protein589198_184-11-1-0-E4-F COVI PTA-2732 Nov. 27, 2000 23 DNA 47-14-1-C3-CL05 APIP 98921 Oct. 15, 1998 24 Protein 47-14-1-C3-CL0 5 APIP 98921 Oct.15, 1998 25 DNA 545542_182-1-2-0-D12-F FGF-22 PTA-2534 Sep. 27, 2000 26Protein 545542_182-1-2-0-D12-F FGF-22 PTA-2534 Sep. 27, 2000 27 DNA117401_106-006-4-0-B11-F Frangiopogen PTA-2534 Sep. 27, 2000 28 Protein117401_106-006-4-0-B11-F Frangiopogen PTA-2534 Sep. 27, 2000 29 DNA133431_105-092-4-0-G11-F Armapoptin PTA-2534 Sep. 27, 2000 30 Protein133431_105-092-4-0-G11-F Armapoptin PTA-2534 Sep. 27, 2000 31 DNA477709_174-8-2-0-C10-F Pretactilin PTA-2534 Sep. 27, 2000 32 Protein477709_174-8-2-0-C10-F Pretactilin PTA-2534 Sep. 27, 2000 33 DNA145606_106-023-2-0-B3-F MS4A5 PTA-2534 Sep. 27, 2000 34 Protein145606_106-023-2-0-B3-F MS4A5 PTA-2534 Sep. 27, 2000 35 DNA1000769575_208-22-1-0-B2- Antaginin PTA-2732 Nov. 27, 2000 F 36 Protein1000769575_208-22-1-0-B2- Antaginin PTA-2732 Nov. 27, 2000 F 37 DNA146994_106-023-4-0-C9-F Beferin PTA-2732 Nov. 27, 2000 38 Protein146994_106-023-4-0-C9-F Beferin PTA-2732 Nov. 27, 2000 39 DNA1000838788_228-28-4-0-F7- RP PTA-2732 Nov. 27, 2000 F 40 Protein1000838788_228-28-4-0-F7- RP PTA-2732 Nov. 27, 2000 F 41 DNA1000943975_160-213-2-0- SSSPI PTA-2732 Nov. 27, 2000 A5-F 42 Protein1000943975_160-213-2-0- SSSPI PTA-2732 Nov. 27, 2000 A5-F 43 DNA147441_106-025-2-0-C11-F CPI-1 44 Protein 147441_106-025-2-0-C11-F CPI-145 DNA 124610_113-003-3-0-H5-F RET-A- PTA-2732 Nov. 27, 2000 MODULIN 46Protein 124610_113-003-3-0-H5-F RET-A- PTA-2732 Nov. 27, 2000 MODULIN 47DNA 1000855165_205-99-1-0-A5- Tifapinix PTA-2732 Nov. 27, 2000 F 48Protein 1000855165_205-99-1-0-A5- Tifapinix PTA-2732 Nov. 27, 2000 F 49DNA 588098_184-11-4-0-H4-F CrypAAT PTA-2732 Nov. 27, 2000 50 Protein588098_184-11-4-0-H4-F CrypAAT PTA-2732 Nov. 27, 2000 51 DNA500721700_204-43-4-0-H10- Tifapinix- F A58S 52 Protein500721700_204-43-4-0-H10- Tifapinix- F A58S 53 DNA789749_182-14-3-0-C12-F Plasminute PTA-2732 Nov. 27, 2000 54 Protein789749_182-14-3-0-C12-F Plasminute PTA-2732 Nov. 27, 2000 55 DNA519757_184-4-2-0-F7-F CALSIGN PTA-2732 Nov. 27, 2000 56 Protein519757_184-4-2-0-F7-F CALSIGN PTA-2732 Nov. 27,2000 57 DNA625004_188-15-4-0-H6-F vCOL16A1 PTA-2534 Sep. 27, 2000 58 Protein625004_188-15-4-0-H6-F vCOL16A1 PTA-2534 Sep. 27, 2000 59 DNA422353_145-11-3-0-E7-F NK5 PTA-2534 Sep. 27, 2000 60 Protein422353_145-11-3-0-E7-F NK5 PTA-2534 Sep. 27, 2000 61 DNA500715621_204-15-3-0-C6- PLasminogen PTA-2534 Sep. 27, 2000 F CarrierProtein (PLCP) 62 Protein 500715621_204-15-3-0-C6- PLasminogen PTA-2534Sep. 27, 2000 F Carrier Protein (PLCP) 63 DNA 165843_116-008-4-0-G4-FNovel PTA-2534 Sep. 27, 2000 Calpastatin 1 (NC1) 64 Protein165843_116-008-4-0-G4-F Novel PTA-2534 Sep. 27, 2000 Calpastatin 1 (NC1)65 DNA 335752_157-15-4-0-B11-F Novel Calpastatin 2 (NC2) 66 Protein335752_157-15-4-0-B11-F Novel Calpastatin 2 (NC2) 67 DNA646607_181-15-2-0-E2-F Benzodiazepine PTA-2534 Sep. 27, 2000 Receptor 2(BZRP-R2) 68 Protein 646607_181-15-2-0-E2-F Benzodiazepine PTA-2534 Sep.27, 2000 Receptor 2 (BZRP-R2) 69 DNA 229654_114-049-1-0-F12-F LAP 70Protein 229654_114-049-1-0-F12-F LAP 71 DNA 338116_174-1-1-0-B10-F ShortPTA-2534 Sep. 27, 2000 Histone Deacetylase (SHDAC) 72 Protein338116_174-1-1-0-B10-F Short PTA-2534 Sep. 27, 2000 Histone Deacetylase(SHDAC) 73 DNA 500716683_204-24-2-0-D12- Protease- PTA-2534 Sep. 27,2000 F associated Paraplegin (PAP) 74 Protein 500716683_204-24-2-0-D12-Protease- PTA-2534 Sep. 27, 2000 F associated Paraplegin (PAP) 75 DNA500760207_205-58-4-0-H6-F Ketothiolase (KT) 76 Protein500760207_205-58-4-0-H6-F Ketothiolase (KT) 77 DNA122421_105-076-4-0-H1-F BASI2 78 Protein 122421_105-076-4-0-H1-F BASI279 DNA 99483_105-016-1-0-D7-F KSPI1 PTA-2534 Sep. 27, 2000 80 Protein99483_105-016-1-0-D7-F KSPI1 PTA-2534 Sep. 27, 2000 81 DNA517778_184-5-3-0-G3-F Amyloid Apoptotic Receptor (AAR) 82 Protein517778_184-5-3-0-G3-F Amyloid Apoptotic Receptor (AAR) 83 DNA100038_105-017-4-0-E4-F Soluble Activator of Wnt 1 (SAW-1) 84 Protein100038_105-017-4-0-E4-F Soluble Activator of Wnt 1 (SAW-1) 85 DNA100523_105-019-1-0-F3-F Soluble Activator of Wnt 1 (SAW-1) 86 Protein100523_105-019-1-0-F3-F Soluble Activator of Wnt 1 (SAW-1) 87 DNA116470_105-063-3-0-H7-F Dopamine AMPhetamine INhibitor (Dampin) 88Protein 116470_105-063-3-0-H7-F Dopamine AMPhetamine INhibitor (Dampin)89 DNA 122600_105-077-3-0-F9-F Dopamine PTA-2732 Nov. 27, 2000AMPhetamine INhibitor (Dampin) 90 Protein 122600_105-077-3-0-F9-FDopamine PTA-2732 Nov. 27, 2000 AMPhetamine INhibitor (Dampin) 91 DNA651658_181-35-2-0-C8-F VAGS 92 Protein 651658_181-35-2-0-C8-F VAGS 93DNA 150011_110-006-3-0-D5-F TFPI-C16Pfs 94 Protein150011_110-006-3-0-D5-F TFPI-C16Pfs 95 DNA 500737461_205-43-3-0-E3-FTFPI- M162Qfs 96 Protein 500737461_205-43-3-0-E3-F TFPI- M162Qfs 97 DNA100545_105-019-2-0-E3-F Soluble Activator of Wnt 2 (SAW-2) 98 Protein100545_105-019-2-0-E3-F Soluble Activator of Wnt 2 (SAW-2) 99 DNA479155_174-4-4-0-C8-F ADEVAR PTA-2732 Nov. 27, 2000 100 Protein479155_174-4-4-0-C8-F ADEVAR PTA-2732 Nov. 27, 2000 101 DNA586587_181-9-2-0-C5-F ATP-binding cassette 1, hABC 102 Protein586587_181-9-2-0-C5-F ATP-binding cassette, hABC 103 DNA620315_188-13-1-0-G12-F MOBP-81h PTA-2534 Sep. 27, 2000 104 Protein620315_188-13-1-0-G12-F MOBP-81h PTA-2534 Sep. 27, 2000 105 DNA646477_181-19-2-0-F4-F novel Apolipoprotein H (NAPOH) 106 Protein646477_181-19-2-0-F4-F novel Apolipoprotein H (NAPOH) 107 DNA113165_105-056-3-0-G12-F human JNK3- binding protein (hJNK3-BP) 108Protein 113165_105-056-3-0-G12-F human JNK3- binding protein (hJNK3-BP)109 DNA 231462_117-065-1-0-G11-F DROCK2 110 Protein231462_117-065-1-0-G11-F DROCK2 111 DNA 500723589_205-34-3-0-G4-F Novel17 beta- hydroxysteroid dehydrogenase type 2 (NBHSD2) 112 Protein500723589_205-34-3-0-G4-F Novel 17 beta- hydroxysteroid dehydrogenasetype 2 (NBHSD2)

TABLE II SEQ ID Signal Mature Polyadenylation NO: ORF Peptide peptideSignal PolyA tail 1 [1435-1836] — — [1965-1970] [2001-2016] 3  [39-917] [39-116] [117-917] [1045-1050] [1066-1081] 5  [84-317]  [84-140][141-317] [397-402] [423-438] 7  [32-748] [32-91]  [92-748] [928-933][953-968] 9 [254-574] [254-295] [296-574] — — 11 [254-574] [254-295][296-574] — — 13 [254-574] [254-295] [296-574] — — 15 [254-574][254-295] [296-574] — — 17  [327-1013] — — [1131-1136] [1160-1175] 19[112-813] [112-162] [163-813] — — 21  [127-1020] [127-183]  [184-1020] —— 23  [10-1212] [10-60]  [61-1212] [1709-1714] [1733-1746] 25 [127-879][127-198] [199-879] — [1224-1239] 27 [116-961] — — [1145-1150][1164-1179] 29  [345-1118] [345-404]  [405-1118] — [1103-1118] 31 [14-1048] [14-91]  [92-1048] [1234-1239] [1258-1273] 33  [73-672] — —[689-694] [708-723] 35 [119-655] — — [809-814] [830-845] 37  [17-259] —— — — 39  [260-1048] [260-319]  [320-1048] [1782-1787] [1801-1816] 41 [91-462]  [91-180] [181-462] [607-612] [628-643] 43 [228-501] [228-326][327-501] — — 45  [98-934] — — — — 47  [267-1139] [267-350]  [351-1139][1246-1251] [1279-1294] 49  [48-1100]  [48-119]  [120-1100] [1159-1164][1179-1194] 51  [290-1162] [290-373]  [374-1162] [1269-1274] [1302-1317]53 [1044-1664] — — [1869-1874] [1892-1907] 55  [26-628] — — [766-771][795-809] 57 [476-964] — — [1101-1106] [1118-1133] 59  [79-642] — —[799-804] [823-838] 61 [159-764] [159-221] [222-764] — — 63 [195-587][195-260] [261-587] [578-583] [604-618] 65 [177-767] [177-242] [243-767][814-819] [822-836] 67  [63-572] — — [750-755] [774-789] 69  [67-2427] [67-114]  [115-2427] [2522-2527] [2541-2556] 71  [8-763]  [8-58] [59-763] [1562-1567] [1588-1603] 73  [9-395]  [9-56]  [57-395] —[864-879] 75  [88-1269] — — [1594-1599] [1619-1634] 77  [69-875] [69-131] [132-875] [1599-1604] [1627-1642] 79  [344-1144] [344-433] [434-1144] — — 81  [27-689]  [27-122] [123-689] [1302-1307] [1325-1406]83 [118-510] [118-189] [190-510] [1718-1723] [1739-1754] 85 [118-510][118-189] [190-510] [1718-1723] [1739-1754] 87 [152-655] — — [1399-1404][1416-1431] 89 [152-655] — — [1399-1404] [1416-1431] 91  [48-1301] [48-119]  [120-1301] [1360-1365] [1402-1417] 93 [278-733] [278-334][335-733] [1072-1077] [1101-1115] 95 [253-744] [253-336] [337-744][1269-1274] [1292-1307] 97 [118-504] [118-189] [190-504] [1819-1824][1840-1855] 99  [95-613] — — [636-641] [652-667] 101 [154-639] — —[1023-1028] [1047-1062] 103 [150-392] — — —  [63-933] 105  [35-1069][35-91]  [92-1069] [1146-1151] [1172-1187] 107  [16-1449] — —[1483-1488] [1505-1520] 109  [95-1252]  [95-139]  [140-1252] [1751-1756][1774-1789] 111  [103-1263] — — [1341-1346] [1365-1408]

TABLE III SEQ ID NO: Positions of immunogenic epitopes 2  21 . . . 28:34 . . . 42: 56 . . . 65: 80 . . . 85: 95 . . . 105: 128 . . . 133 4  32. . . 39: 57 . . . 66: 78 . . . 84: 92 . . . 105: 152 . . . 157: 165 . .. 171: 262 . . . 270: 277 . . . 287 6  23 . . . 33: 34 . . . 41: 49 . .. 63 8  42 . . . 48: 53 . . . 69: 76 . . . 94: 145 . . . 154: 165 . . .171: 179 . . . 188: 186 . . . 200: 229 . . . 238 10  11 . . . 20: 36 . .. 55: 63 . . . 70: 79 . . . 94 12  11 . . . 20: 36 . . . 55: 63 . . .70: 79 . . . 94 14  11 . . . 20: 36 . . . 55: 63 . . . 70: 79 . . . 9416  11 . . . 20: 36 . . . 55: 63 . . . 70: 79 . . . 94 18  10 . . . 22:80 . . . 91: 100 . . . 110: 122 . . . 128: 134 . . . 141: 151 . . . 162:160 . . . 173: 191 . . . 202: 216 . . . 227 20  21 . . . 28: 54 . . .62: 70 . . . 81: 83 . . . 91: 95 . . 101: 110 . . . 124: 134 . . . 139:180 . . . 190 22  20 . . . 29: 33 . . . 39: 43 . . . 53: 82 . . . 92:253 . . . 264 24  16 . . . 27: 87 . . . 97: 152 . . . 159: 169 . . .175: 178 . . . 188: 213 . . . 221: 273 . . . 282: 308 . . . 313: 339 . .. 347: 385 . . . 395 26  45 . . . 55: 52 . . . 63: 106 . . . 117: 118 .. . 128: 126 . . . 131: 148 . . . 155: 157 . . . 164: 172 . . . 190: 212. . . 221: 232 . . . 247 28  44 . . . 53: 55 . . . 65: 82 . . . 90: 93 .. . 114: 119 . . . 132: 148 . . . 163: 174 . . . 179: 176 . . . 181: 199. . . 219: 218 . . . 228: 242 . . . 253: 272 . . . 278 30  1 . . . 6: 41. . . 46: 92 . . . 102: 133 . . . 139: 143 . . . 163: 161 . . . 181: 185. . . 195: 214 . . . 221 32  53 . . . 77: 120 . . . 130: 144 . . . 159:159 . . . 169: 196 . . . 202: 266 . . . 272: 331 . . . 344 34 147 . . .157: 189 . . . 199 36 113 . . . 125: 139 . . . 151: 149 . . . 160 38  1. . . 8: 49 . . . 63: 66 . . . 76 40  27 . . . 35: 106 . . . 111: 183 .. . 194: 222 . . . 228: 241 . . . 247: 255 . . . 262 42  38 . . . 49: 49. . . 54: 71 . . . 82: 92 . . . 116 44  1 . . . 19 46  1 . . . 8: 9 . .. 14: 70 . . . 80: 85 . . . 92: 110 . . . 116: 145 . . . 158: 202 . . .216: 231 . . . 246: 244 . . . 253: 262 . . . 276 48  57 . . . 63: 85 . .. 96: 104 . . . 111: 114 . . . 121: 127 . . . 142: 159 . . . 169: 169 .. . 178: 185 . . . 191: 206 . . . 214: 213 . . . 222: 228 . . . 250 50 58 . . . 67: 116 . . . 125: 149 . . . 154: 188 . . . 193: 213 . . .218: 233 . . . 241: 332 . . . 339 52  56 . . . 63: 85 . . . 96: 104 . .. 111: 114 . . . 121: 127 . . . 142: 159 . . . 169: 169 . . . 178: 185 .. . 191: 206 . . . 214: 213 . . . 222: 228 . . . 250 54  21 . . . 30:124 . . . 137: 147 . . . 159: 181 . . . 189 56  55 . . . 64: 80 . . .86: 167 . . . 174 58  3 . . . 15: 12 . . . 42: 40 . . . 66: 75 . . . 85:90 . . . 107: 123 . . . 142: 147 . . . 159 60  30 . . . 39: 73 . . . 89:96 . . . 102: 163 . . . 187 62  20 . . . 31: 89 . . . 101: 106 . . .116: 157 . . . 172: 180 . . . 194 64  28 . . . 34: 37 . . . 45: 49 . . .61: 61 . . . 77: 102 . . . 108 66  27 . . . 35: 37 . . . 45: 49 . . .61: 61 . . . 77: 102 . . . 109: 144 . . . 152: 170 . . . 180: 179 . . .188 68  22 . . . 36: 151 . . . 156: 161 . . . 169 70  19 . . . 24: 34 .. . 45: 79 . . . 94: 100 . . . 107: 146 . . . 152: 161 . . . 168: 174 .. . 179: 199 . . . 204: 238 . . . 246: 259 . . . 269: 329 . . . 342: 380. . . 393: 390 . . . 395: 393 . . . 398: 395 . . . 400: 397 . . . 404:408 . . . 414: 427 . . . 434: 447 . . . 456: 461 . . . 474: 481 . . .489: 492 . . . 499: 506 . . . 513: 520 . . . 540: 556 . . . 563: 561 . .. 568: 584 . . . 590: 596 . . . 604: 626 . . . 632: 629 . . . 634: 634 .. . 656: 654 . . . 659: 675 . . . 681: 714 . . . 731: 730 . . . 743: 745. . . 766: 768 . . . 786 72  97 . . . 110: 234 . . . 245 74  10 . . .23: 27 . . . 32: 33 . . . 44: 103 . . . 108: 111 . . . 122 76 116 . . .122: 182 . . . 188: 205 . . . 215: 223 . . . 231: 234 . . . 241: 351 . .. 356: 364 . . . 374 78  67 . . . 73: 71 . . . 85: 142 . . . 148: 176 .. . 195: 229 . . . 237: 236 . . . 246: 248 . . . 268 80  25 . . . 44: 54. . . 61: 93 . . . 99: 99 . . . 108: 107 . . . 123: 129 . . . 144: 164 .. . 172: 176 . . . 185: 203 . . . 210: 214 . . . 221: 225 . . . 233: 243. . . 253 82  42 . . . 48: 84 . . . 93: 104 . . . 118: 122 . . . 132:141 . . . 147: 153 . . . 161 84  42 . . . 51: 76 . . . 94: 97 . . . 12686  42 . . . 51: 76 . . . 94: 97 . . . 126 88  6 . . . 14: 13 . . . 23:25 . . . 39: 36 . . . 42: 59 . . . 67: 79 . . . 86: 110 . . . 120: 123 .. . 132: 133 . . . 145: 153 . . . 167 90  6 . . . 14: 13 . . . 23: 25 .. . 39: 36 . . . 42: 59 . . . 67: 79 . . . 86: 110 . . . 120: 123 . . .132: 133 . . . 145: 153 . . . 167 92  25 . . . 33: 31 . . . 48: 65 . . .73: 125 . . . 134: 183 . . . 192: 216 . . . 221: 255 . . . 260: 280 . .. 285: 300 . . . 308: 400 . . . 405 94  48 . . . 54: 76 . . . 87: 95 . .. 102: 107 . . . 115: 118 . . . 125: 131 . . . 141 96  57 . . . 63: 85 .. . 96: 104 . . . 111: 116 . . . 124: 127 . . . 134: 140 . . . 155 98 42 . . . 51: 76 . . . 94: 116 . . . 123 100  26 . . . 33: 46 . . . 54:104 . . . 117: 125 . . . 130 102  15 . . . 23: 44 . . . 55: 52 . . . 62:77 . . . 83: 83 . . . 88: 115 . . . 124: 132 . . . 148: 145 . . . 156104  2 . . . 23: 34 . . . 39: 41 . . . 46: 50 . . . 60: 67 . . . 80 106 21 . . . 30: 40 . . . 50: 49 . . . 62: 99 . . . 106: 123 . . . 133: 156. . . 169: 189 . . . 198: 197 . . . 205: 203 . . . 216: 224 . . . 232:232 . . . 246: 300 . . . 315: 336 . . . 344 108  9 . . . 20: 33 . . .52: 68 . . . 75: 91 . . . 97: 123 . . . 130: 175 . . . 189: 186 . . .193: 195 . . . 204: 216 . . . 227: 229 . . . 234: 246 . . . 252: 249 . .. 254: 302 . . . 320: 386 . . . 396: 402 . . . 412: 409 . . . 415: 429 .. . 451 110  8 . . . 17: 70 . . . 78: 111 . . . 123: 142 . . . 155: 176. . . 191: 189 . . . 194: 191 . . . 198: 206 . . . 220: 235 . . . 240:250 . . . 262: 285 . . . 291: 331 . . . 340: 346 . . . 355 112  25 . . .35: 115 . . . 131: 207 . . . 214: 230 . . . 235: 272 . . . 278: 291 . .. 298: 313 . . . 318: 336 . . . 345: 362 . . . 374: 377 386

TABLE IV SEQ ID NO: Preferentially excluded fragments Preferentiallyincluded fragments 1 [1-507]; [1524-2004] [508-1523]; [2005-2016] 3[1-477]; [507-849]; [851-1081] [478-506]; [850-850] 5 [1-430] [431-438]7 [1-816] [817-968] 9 [1-190]; [205-336]; [338-527] [191-204];[337-337]; [528-730] 11 [1-190]; [205-336]; [338-527] [191-204];[337-337]; [528-733] 13 [1-190]; [205-336]; [338-527] [191-204];[337-337]; [528-732] 15 [1-190]; [205-336]; [338-527] [191-204];[337-337]; [528-733] 17 [31-415]; [417-476] [1-30]; [416-416];[477-1175] 19 [1-239]; [241-593]; [673-732] [240-240]; [594-672];[733-844] 21 [1-533]; [1323-1455]; [1459-1751] [534-1322]; [1456-1458];[1752-1997] 23 [1-289]; [291-320] [290-290]; [321-1746] 25 [1-528][529-1239] 27 [1-417]; [814-1162] [418-813]; [1163-1179] 29 [1-172];[178-334] [173-177]; [335-1118] 31 [1-122]; [385-435] [123-384];[436-1816] 33 [1-585] [586-643] 35 [1-436]; [444-487] [437-443];[488-501] 37 [1-71]; [73-466] [72-72]; [467-845] 39 [1-500] [501-517] 41[1-575]; [683-1045]; [576-682]; [1046-1046]; [1047-1141]; [1149-1178][1142-1148]; [1179-1194] 43 [1-558] [559-960] 45 [1-5 10]; [533-572][511-532]; [573-1294] 47 [1-5 19]; [523-552] [520-522]; [553-1273] 49[1-723] None 51 [1-533]; [556-595] [534-555]; [596-1317] 53 [1-64];[67-441]; [1035-1306]; [65-66]; [442-1034]; [1307-1405]; [1406-1488];[1514-1711]; [1489-1513]; [1712-1712]; [1713-1787]; [1789-1892][1788-1788]; [1893-1907] 55 [1-483] [484-809] 57 [1-494] [495-1133] 59[2-523] [1-1]; [524-838] 61 [1-427] [428-862] 63 [1-30]; [125-299];[301-570] [31-124]; [300-300]; [571-618] 65 [14-105] [1-13]; [106-836]67 [1-293]; [304-541] [294-303]; [542-789] 69 [1-466]; [900-974][467-899]; [975-2556] 71 [1-486]; [526-560]; [987-1588] [487-525];[561-986]; [1589-1603] 73 [1-435]; [486-517]; [599-708]; [436-485];[518-598]; [709-727]; [728-803]; [812-879] [804-811] 75 [1-465][466-1634] 77 [2-394]; [396-564]; [68 1-832]; [1-1]; [395-395];[565-680]; [1207-1294] [833-1206]; [1295-1642] 79 [1-218]; [220-591];[605-663] [219-219]; [592-604]; [664-1466] 81 [1-432] [433-1406] 83[1-339] [340-1754] 85 [1-339] [340-1754] 87 [1-433]; [1261-1355][434-1260]; [1356-1431] 89 [1-433]; [1261-1355] [434-1260]; [1356-1431]91 [1-738]; [884-1342]; [1350-1380] [739-883]; [1343-1349]; [1381-1417]93 [1-494]; [517-581] [495-516]; [582-1115] 95 [1-189]; [191-496];[519-583] [190-190]; [497-518]; [584-1307] 97 [1-339] [340-1855] 99[1-405]; [426-457] [406-425]; [458-667] 101 [1-44]; [666-753];[783-813]; [45-665]; [754-782]; [814-898]; [899-965]; [98 1-1013][966-980]; [1014-1062] 103 [1-77]; [79-412]; [418-456]; [758-916][78-78]; [413-417]; [457-757]; [917-933] 105 [1-287]; [289-635][288-288]; [636-1187] 107 [1-501]; [680-719]; [721-816]; [502-679];[720-720]; [817-821]; [822-853]; [982-1180]; [854-981]; [1181-1181];[1182-1235]; [1237-1383]; [1236-1236]; [1384-1403] [1404-1520] 109[1-393]; [409-503] [394-408]; [504-1789] 111 [1-777]; [779-860];[1365-1408] [778-778]; [861-1364]

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent specification referenced in thisapplication are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

1. A Plasminute polypeptide encoded by an isolated polynucleotide consisting of an open reading frame of the human cDNA of deposited clone 789749_(—)182-14-3-0-C12-F.
 2. An isolated Plasminute protease consisting of amino acids 1 to 207 of SEQ ID NO:54.
 3. The protease of claim 2, wherein said protease is a serine protease.
 4. A composition comprising the protease of claim 2 and a physiologically acceptable carrier.
 5. A composition comprising the polypeptide of claim 3 and a physiologically acceptable carrier.
 6. A composition comprising the polypeptide of claim 1 and a physiologically acceptable carrier.
 7. A method of digesting a protein consisting of contacting a protein with a) a Plasminute polypeptide encoded by an isolated polynucleotide comprising an open reading frame of the human cDNA of deposited clone 789749_(—)182-14-3-0-C12-F; b) a Plasminute protease consisting of amino acids 1 to 207 of SEQ ID NO:54; or c) compositions thereof under conditions that allow for the digestion of said protein.
 8. The method of claim 7, wherein a protein is contacted with a Plasminute polypeptide encoded by an isolated polynucleotide consisting of an open reading frame of the human cDNA of deposited clone 789749_(—)182-14-3-0-C12-F.
 9. The method of claim 7, wherein a protein is contacted with a Plasminute protease consisting of amino acids 1 to 207 of SEQ ID NO:54.
 10. The method of claim 7, wherein a protein is contacted with a composition comprising a carrier and a Plasminute protease consisting of amino acids 1 to 207 of SEQ ID NO:54.
 11. The method of claim 7, wherein a protein is contacted with a composition consisting of a carrier and a Plasminute polypeptide encoded by an isolated polynucleotide consisting of an open reading frame of the human cDNA of deposited clone 789749_(—)182-14-3-0-C12-F. 